Ligf-type systems for bioconversion of lignin-derived compounds

ABSTRACT

The teachings provided herein are generally directed to a method of converting lignin-derived compounds to valuable aromatic chemicals using an enzymatic, bioconversion process. The teachings provide a selection of (i) host cells that are tolerant to the toxic compounds present in lignin fractions; (ii) polypeptides that can be used as enzymes in the bioconversion of the lignin fractions to the aromatic chemical products; (iii) polynucleotides that can be used to transform the host cells to express the selection of polypeptides as enzymes in the bioconversion of the lignin fractions; and (iv) the transformants that express the enzymes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Nos.61/403,440, filed Sep. 15, 2010; and 61/455,709, filed Oct. 25, 2010;each application of which is hereby incorporated herein by reference init's entirety,

SEQUENCE LISTING

The instant application is filed with an ASCII compliant text file of aSequence Listing. The name of the attached file isALIGP004US01_SEQLIST_AS-FILED.txt, and the file was created Aug. 29,2011, is 813 KB in size, and is hereby incorporated herein by referencein its entirety. Because the ASCII compliant text file serves as boththe paper copy required by §1.821(c) and the CRF required by §1.821(e),the statement indicating that the paper copy and CRF copy of thesequence listing are identical is no longer necessary under 37 C.F.R.§1.821(f), as per Federal Register/Vol. 74, No. 206/Tuesday, Oct. 27,2009, Section I.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The teachings provided herein are generally directed to a method ofconverting lignin-derived compounds to valuable aromatic chemicals usingan enzymatic, bioconversion process.

2. Description of the Related Art

Currently, there is a worldwide, global dependence on petroleum as adeplete-able feedstock for the manufacture of fuels and chemicals. Theproblems of using petroleum are so well-known and documented thatthey've become nearly a cliché to the world population. In short,petroleum-based processes are dirty and hazardous. Environmental effectsassociated with the use of petroleum are known to include, for example,air pollution, global warming, damage from extraction, oil spills,tarballs, and health hazards to humans, domestic animals, and wildlife.

Oil refineries, for example, are petroleum-based processes thatprimarily produce gasoline. However, they are also used extensively toproduce valuable and less well-known chemical products used in themanufacture of pharmaceuticals, agrochemicals, food ingredients, andplastics. A clean, green alternative to this market area would beappreciated worldwide.

Bioprocesses can present a clean, green alternative to thepetroleum-based processes, a bioprocess being one that uses organisms,cells, organelles, or enzymes to carry out a commercial process.Biorefineries, for example, can produce, for example, chemicals, heatand power, as well as food, feed, fuel and industrial chemical products.Examples of biorefineries can include wet and dry corn mills, pulp andpaper mills, and the biofuels industry. In leather tanning, hides aresoftened and hair is removed using proteases. In brewing, amylases areused in germinating barley. In cheese-making, rennin is used tocoagulated the proteins in mil. The biofuels industry, for example, hasbeen a point of focus recently, naturally focusing on fuel products toreplace petroleum-based fuels and, as a result, has not developed othervaluable chemical products that also rely on petroleum-based processes.

As such, biorefineries use enzymes to convert natural products to usefulchemicals. A natural product, such as the wood that is used in a pulpand paper mill, contains cellulose, hemicelluloses, and lignin. Atypical range of compositions for a hardwood may be about 40-44%cellulose, about 15-35% hemicelluloses, and about 18-25% lignin.Likewise, a typical range of compositions for a softwood may be about40-44% cellulose, about 20-32% hemicelluloses, and about 25-35% lignin.Since all biofuels come from cellulosic biorefineries, where the key rawmaterial is glucose, derived from cellulose, lignin remainsunderutilized. Lignin is the single most abundant source of aromaticcompounds in nature, and the use of lignin is currently limited to lowvalue applications, such as combustion to generate process heat andenergy for the biorefinery facilities. In the alternative, lignin issold as a natural component of animal feeds or fertilizers.Interestingly, however, lignin is the only plant biomass component basedon aromatic core structures, and such core structures are valuable inthe production of industrial chemicals. One of skill will appreciatethat, unfortunately, a major problem to such a use of lignin remains:the aromatic compounds present in the lignin fraction of a biorefineryinclude toxic compounds that inhibit the growth and survival ofindustrial microbes. For at least these reasons, processes forconverting lignin fractions to industrial products using industrialmicrobes have not been successful.

In view of the above, one of skill will appreciate (i) a clean, greenreplacement for petroleum-based processes in the production of valuablechemical products that include major markets such as, for example,pharmaceuticals, agrochemicals, food ingredients, and plastics; (ii) aprofitable use of the abundant and renewable natural resource availablein lignin, which is currently an industrial waste stream that isunderutilized as an industrial feedstock; (iii) a selection of hostcells that are tolerant to the toxic compounds present in ligninfractions in the feedstock; (iv) a selection of polypeptides that can beused as enzymes in the bioconversion of the lignin fractions to thevaluable chemical products; (v) a selection of polynucleotides that canbe used to transform host cells to express the selection of polypeptidesin the bioconversion of the lignin fractions to the valuable chemicalproducts; (vi) systems that include transformants that express theenzymes, where the transformants can be used to (a) express the enzymeswhile in direct contact with the lignin fractions or (b) express theenzymes for extraction from the cells, after which the extracted enzymesare used directly in contact with the lignin fractions; and (vii) aclean-and-green method of producing valuable chemical products at higherprofits than petroleum-based processes.

SUMMARY

This invention is generally directed to a recombinant method ofproducing enzymes for use in the bioconversion of lignin-derivedcompounds to valuable aromatic chemicals. In some embodiments, theteachings are directed to an isolated recombinant polypeptide,comprising an amino acid sequence having at least 95% identity to SEQ IDNO:101. The sequence can conserve residues T19, I20, S21, P22, V24, W25,T27, K28, Y29, A30, H33, K34, G35, F36, D39, I40, V41, P42, G43, G44,F45, G47, I48, E50, R51, T52, G53, G54, K100, A101, N104, V111, G112,M115, F116, P166, W107, Y184, Y187, R188, G191, G192, and F195.

In some embodiments, the teachings are directed to an isolatedrecombinant polypeptide, comprising SEQ ID NO:101; or conservativesubstitutions thereof outside of the conserved residues. The conservedresidues can include T19, I20, S21, P22, V24, W25, T27, K28, Y29, A30,H33, K34, G35, F36, D39, I40, V41, P42, G43, G44, F45, G47, I48, E50,R51, T52, G53, G54; K100, A101, N104, V111, G112, M115, F116, P166,W107, Y184, Y187, R188, G191, G192, and F195.

In some embodiments, the teachings are directed to an isolatedrecombinant glutathione S-transferase enzyme, comprising an amino acidsequence having at least 95% identity to SEQ ID NO:101. The amino acidsequence can conserve residues T19, I20, S21, P22, V24, W25, T27, K28,Y29, A30, H33, K34, G35, F36, D39, I40, V41, P42, G43, G44, F45, G47,I48, E50, R51, T52, G53, G54; K100, A101, N104, V111, G112, M115, F116,P166, W107, Y184, Y187, R188, G191, G192, and F195; wherein, the aminoacid sequence functions to cleave a beta-aryl ether.

In some embodiments, the teachings are directed to an isolatedrecombinant glutathione S-transferase enzyme, comprising an amino acidsequence having at least 95% identity to SEQ ID NO:101; wherein, theamino acid sequence functions to cleave a beta-aryl ether.

In some embodiments, the teachings are directed to an isolatedrecombinant polypeptide, comprising (i) a length ranging from about 279to about 281 amino acids; (ii) a first amino acid region consisting ofresidues 19-54 from SEQ ID NO:101, or conservative substitutions thereofoutside of conserved residues T19, I20, S21, P22, V24, W25, T27, K28,Y29, A30, H33, K34, G35, F36, D39, I40, V41, P42, G43, G44, F45, G47,I48, E50, R51, T52, G53, and G54; wherein, the first amino acid regioncan be located in the recombinant polypeptide from about residue 14 toabout residue 59; and, (iii) a second amino acid region consisting ofresidues 98-221 from SEQ ID NO:101, or conservative substitutionsthereof outside of conserved residues K100, A101, N104, V111, G112,M115, F116, P166, W107, Y184, Y187, R188, G191, G192, and F195; wherein,the second amino acid region is located in the recombinant polypeptidefrom about residue 93 to about residue 226.

In some embodiments, the teachings are directed to an isolatedrecombinant glutathione S-transferase enzyme, comprising (i) a lengthranging from about 279 to about 281 amino acids; (ii) a first amino acidregion having at least 95% identity to residues 19-54 from SEQ ID NO:101while conserving residues T19, I20, S21, P22, V24, W25, T27, K28, Y29,A30, H33, K34, G35, F36, D39, I40, V41, P42, G43, G44, F45, G47, I48,E50, R51, T52, G53, and G54; wherein, the first amino acid region islocated in the recombinant polypeptide from about residue 14 to aboutresidue 59; and, (iii) a second amino acid region having at least 95%identity to residues 98-221 from SEQ ID NO:101 while conserving residuesK100, A101, N104, V111, G112, M115, F116, P166, W107, Y184, Y187, R188,G191, G192, and F195; wherein, the second amino acid region can belocated in the recombinant polypeptide from about residue 93 to aboutresidue 226; and, the recombinant glutathione S-transferase enzyme canfunction to cleave a beta-aryl ether.

In some embodiments, the teachings are directed to an isolatedrecombinant glutathione S-transferase enzyme, comprising an amino acidsequence having at least 95% identity to SEQ ID NO:541; wherein, theamino acid sequence functions to cleave a beta-aryl ether.

In some embodiments, the teachings are directed to an isolatedrecombinant polypeptide, comprising (i) a length ranging from about 256to about 260 amino acids; (ii) a first amino acid region consisting ofresidues 47-57 from SEQ ID NO:541, or conservative substitutions thereofoutside of conserved residues A47, I48, N49, P50, G52, V54, P55, V56,L57; wherein, the first amino acid region is located in the recombinantpolypeptide from about residue 45 to about residue 57; (iii) a secondamino acid region consisting of 63-76 from SEQ ID NO:541; and, (iv) athird amino acid region consisting of residues 99-230 from SEQ IDNO:541, or conservative substitutions thereof outside of conservedresidues R100, Y101, K104, D107, M111, N112, S115, M116, K176, L194,I197, N198, S201, H202, and M206; wherein, the second amino acid regionis located in the recombinant polypeptide from about residue 94 to aboutresidue 235.

In some embodiments, the teachings are directed to an isolatedrecombinant glutathione S-transferase enzyme, comprising (i) a lengthranging from about 279 to about 281 amino acids; (ii) a first amino acidregion having at least 95% identity to 47-57 from SEQ ID NO:541, orconservative substitutions thereof outside of conserved residues A47,I48, N49, P50, G52, V54, P55, V56, L57; wherein, the first amino acidregion can be located in the recombinant polypeptide from about residue45 to about residue 57; (iii) a second amino acid region consisting of63-76 from SEQ ID NO:541; and, (iv) a third amino acid region having atleast 95% identity to residues 99-230 from SEQ ID NO:541, orconservative substitutions thereof outside of conserved residues R100,Y101, K104, D107, M111, N112, S115, M116, K176, L194, I197, N198, S201,H202, and M206; wherein, the second amino acid region can be located inthe recombinant polypeptide from about residue 94 to about residue 235;wherein, the recombinant glutathione S-transferase enzyme functions tocleave a beta-aryl ether.

In some embodiments, an amino acid substitution outside of the conservedresidues can be a conservative substitution. And, in many embodiments,the amino acid sequence can function to cleave a beta-aryl ether.

The teachings are also directed to a method of cleaving a beta-arylether bond, the comprising contacting a polypeptide taught herein with alignin-derived compound having (i) a beta-aryl ether bond and (ii) amolecular weight ranging from about 180 Daltons to about 3000 Daltons;wherein, the contacting occurs in a solvent environment in which thelignin-derived compound is soluble.

In some embodiments, the lignin-derived compound has a molecular weightof about 180 Daltons to about 1000 Daltons. In some embodiments, thesolvent environment comprises water. And, in some embodiments, thesolvent environment comprises a polar organic solvent.

The teachings are also directed to a system for bioprocessinglignin-derived compounds, the system comprising a polypeptide taughtherein, a lignin-derived compound having a beta-aryl ether bond and amolecular weight ranging from about 180 Daltons to about 3000 Daltons;and, a solvent in which the lignin-derived compound is soluble; wherein,the system functions to cleave the beta-aryl ether bond by contactingthe polypeptide with the lignin-derived compound in the solvent.

The teachings are also directed to a recombinant polynucleotidecomprising a nucleotide sequence that encodes a polypeptide taughtherein. Likewise, the teachings are also directed to a vector or plasmidcomprising the polynucleotide, as well as a host cell transformed by thevector or plasmid to express the polypeptide.

The teachings are also directed to a method of cleaving a beta-arylether bond, the method comprising (i) culturing a host cell taughtherein under conditions suitable to produce a polypeptide taught herein;(ii) recovering the polypeptide from the host cell culture; and, (iii)contacting the polypeptide of claim 1 with a lignin-derived compoundhaving a beta-aryl ether bond and a molecular weight ranging from about180 Daltons to about 3000 Daltons; wherein, the contacting occurs in asolvent environment in which the lignin-derived compound is soluble.

In some embodiments, the host cell can be E. Coli or an Azotobacterstrain, such as Azotobacter vinelandii. And, in some embodiments, thelignin-derived compound can have a molecular weight of about 180 Daltonsto about 1000 Daltons.

The teachings are also directed to a system for bioprocessinglignin-derived compounds, the system comprising (i) a transformed hostcell taught herein; (ii) a lignin-derived compound having a beta-arylether bond and a molecular weight ranging from about 180 Daltons toabout 3000 Daltons; and, (iii) a solvent in which the lignin-derivedcompound is soluble; wherein, the system functions to cleave thebeta-aryl ether bond by contacting a polypeptide taught herein with thelignin-derived compound in the solvent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B illustrate general concepts of the biorefinery anddiscovery processes discussed herein, according to some embodiments.

FIG. 2 illustrates the structures of some building block chemicals thatcan be produced using bioconversions, according to some embodiments.

FIG. 3 is an example of a beta-etherase catalyzed hydrolysis of a modellignin dimer, α-O-(β-methylumbelliferyl) acetovanillone (MUAV),according to some embodiments.

FIG. 4 illustrates unexpected results from biochemical activity assaysfor beta-etherase function for the S. paucimobilis positive controlpolypeptides, and the N. aromaticivorans putative beta-etherasepolypeptide, according to some embodiments.

FIG. 5 illustrates beta-aryl-ether compounds to be tested as substratesrepresenting native lignin structures, according to some embodiments.

FIG. 6 illustrates pathways of guaiacylglycerol-β-guaiacyl ether (GGE)metabolism by S. paucimobilis, according to some embodiments.

FIG. 7 illustrates an example of a biochemical process for theproduction of catechol from lignin oligomers, according to someembodiments.

FIG. 8 illustrates an example of a biochemical process for theproduction of vanillin from lignin oligomers, according to someembodiments.

FIG. 9 illustrates an example of a biochemical process for theproduction of 2,4-diaminotoluene from lignin oligomers, according tosome embodiments.

FIG. 10 illustrates process schemes for additional product targets thatinclude ortho-cresol, salicylic acid, and aminosalicylic acid, for theproduction of valuable chemicals from lignin oligomers, according tosome embodiments.

DETAILED DESCRIPTION OF THE INVENTION

This invention is generally directed to a recombinant method ofproducing enzymes for use in the bioconversion of lignin-derivedcompounds to valuable aromatic chemicals. Currently, the art is limitedin it's ability to control the degradation of lignin to produce usefulproducts, as it's limited in it's knowledge of enzymes that are capableof selectively converting lignin into desired aromatic compounds.Generally, the art knows two basic things: (1) lignin is complex; and(2) bacterial lignin degradation systems are therefore at least ascomplex as lignin itself. Accordingly, and for at least these reasons,the teachings provided herein offer a valuable, unexpected, andsurprising set of systems, methods, and compositions of matter that willbe useful in the production of industrially useful aromatic chemicals.

FIGS. 1A and 1B illustrate general concepts of the biorefinery anddiscovery processes discussed herein, according to some embodiments.FIG. 1A shows a generalized example of a use of recombinant microbialstrains in biotransformations for the production of aromatic chemicalsfrom lignin-derived compounds. Biorefinery process 100 converts asoluble biorefinery lignin 105 through a series of biotransformationsusing a transformed host cell. The biorefinery lignin 105 is a feedstockcomprising a lignin-derived compound which can be, for example, acombination of lignin-derived monomers and oligomers. “Biotransformation1” 107 can be used to selectively cleave a bond on or between monomersto create additional lignin monomers 110. “Biotransformation 2” 112 canbe used to selectively cleave an additional bond on or between monomersto create mono-aromatic commercial products 115. FIG. 1B shows adiscovery process 120, which includes selecting a host cell strain thatis tolerant to toxic lignin-derived compounds. The strain acquisition125 includes growth of the strain, sample preparation, and storage. Aset of bacterial strains are obtained for testing strain tolerance tosoluble biorefinery lignin samples.

In some embodiments, the strains can be selected for (i) havingwell-characterized aromatic and xenobiotic metabolisms; (ii) annotatedgenome sequences; and (iii) prior use in fermentation processes at pilotor larger scales. Examples of strains can include, but are not limitedto, Azotobacter vinelandii (ATCC BAA-1303 DJ), Azotobacter chroococcum(ATCC 4412 (EB Fred) X-50), Pseudomonas putida (ATCC BAA-477 Pf-5),Pseudomonas fluorescens (ATCC 29837 NCTC 1100). Stains can be streakedon relevant rich media plates as described by the accompanying ATCCliterature for revival. Individual colonies (5 each) can be picked andcultured on relevant liquid media to saturation. Culture samplesprepared in a final glycerol concentration of 12.5% can be flash-frozenand stored at −80° C.

The model substrate synthesis 150 for use in the biochemical screeningfor selective activity can be outsourced through a contract researchorganization (CRO). The enzyme discovery effort can initially be focusedon identifying potential beta-etherase candidate genes identifiedthrough bioinformatic methods. The identification of candidates havingbeta-etherase activity is the 1st step towards generating ligninmonomers from lignin oligomers present in soluble lignin streams. Thefluorescent substrate α-O-(β-methylumbelliferyl) acetovanillone (MUAV),for example, can be used in in vitro assays to identify beta-etherasefunction (Acme Biosciences, Mt. View, Calif.). The formation of 4methylumbelliferone (4MU) upon hydrolysis of the aryl ether bond can bemonitored by fluorescence, for example, at λex=365 nm and λem=450 nm (or460 nm).

The gene synthesis, cloning, and transformation step 145 can includecombining bioinformatic methods with known information about enzymesshowing a desired, selective enzyme activity. For example,bioinformatics can produce a putative beta-etherase sequence that sharesa significant homology to the S. paucimobilis ligE and ligFbeta-etherase sequences. See Masai, E., et al. Journal of Bacteriology(3):1768-1775 (2003)(“Masai”), which is hereby incorporated herein init's entirety by reference. The S. paucimobilis sequences can be used aspositive controls for biochemical assays to show relative activities inan enzyme discovery strategy.

The gene synthesis, cloning, and transformation step 145 can beperformed using any method known to one of skill. For example, all genescan be synthesized directly as open reading frames (ORFs) fromoligonucleotides by using standard PCR-based assembly methods, and usingthe E. coli codon bias. The end sequences can contain adaptors (BamHIand HindIII) for restriction digestion and cloning into the E. coliexpression vector pET24a (Novagen). Internal BamHI and HindIII sites canbe excluded from the ORF sequences during design of theoligonucleotides. Assembled genes can be cloned into the proprietarycloning vector (pGOV4), transformed into E. coli CH₃ chemicallycompetent cells, and DNA sequences determined (Tocore Inc.) frompurified plasmid DNA. After sequence verification, restriction digestioncan be used to excise each ORF fragment from the cloning vector, and thesequence can be sub-cloned into pET24a. The entire set of ligE and ligFbearing plasmids can then be transformed into E. coli BL21 (DE3) whichcan serve as the host strain for beta-etherase expression andbiochemical testing.

The enzyme screening 155 is done to identify novel etherases 160. Thefluorescent substrate MUAV can be used to screen for and identifybeta-etherase activity from the recombinant E. coli clones. Expressionof the beta-etherase genes can be done in 5 ml or 25 ml samples of therecombinant E. coli strains in LB medium using induction with IPTG.Following induction, and cell harvest, cell pellets can be lysed usingthe BPER (Invitrogen) cell lysis system. Cell extracts can be tested inthe in vitro biochemical assay for beta-etherase activity on thefluorescent substrate MUAV. The formation of 4 methylumbelliferone (4MU)upon hydrolysis of the aryl ether bond in MUAV can be monitored byfluorescence at λex=365 nm and λem=460 nm, and can provide quantitativemeasurement of beta-etherase function. Cell extracts of E. colitransformed with the S. paucimobilis ligE and ligF genes can be theassay positive controls. Test or unknown samples can include, forexample, E. coli strains expressing putative beta-etherase genes from N.aromaticovorans.

The lignin stream acquisition 130 includes a waste lignin stream from abiorefinery for testing. A preliminary characterization of one source ofsuch lignin has shown an aromatic monomer concentration of less than 1g/L and an oligomer concentration of ˜10 g/L. Oligomers appear to beassociated with carbohydrates in 10:1 ratio for sugar:phenolics. Someinformation exists on compounds in the liquid stream, including benzoicacid, vanillin, syringic acid and ferulics, which are routinelyquantified in soluble samples. An average molecular weight of ˜280 hasbeen established for the monomers; and the oligomeric components remainto be characterized.

The strain tolerance testing 135 Strain tolerance will be determined bycell growth upon exposure to biorefinery lignin. Tolerance to thephenolic compounds in biorefinery lignin waste stream will be criticallyimportant to the bioprocess efficiency and high level production ofaromatic chemicals by microbial systems. Cell growth will be quantifiedas a function of respiration by the reduction of soluble tetrazoliumsalts. XTT(2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilideinner salt, Sigma) is reduced to a soluble purple formazan compound byrespiring cells. The formazan product will be detected and quantified byabsorbance at 450 nm.

Strain tolerance testing 135 on soluble lignin can be done in liquidformat in 48 well plates, for example. Each strain can be tested inreplicates of 8, for example, and E. coli can be used as a negativecontrol strain. Strains can first be grown in rich medium to saturation,washed, and OD600 nm of the cultures determined. Equal numbers ofbacteria can be inoculated into wells of the 48-well growth platecontaining minimal medium excluding a carbon source. Increasingconcentrations of soluble lignin fractions, in addition to aminus-lignin positive control, can be added to the wells containing eachspecies to a final volume of 0.8 ml. A benzoic acid content analysis ofthe lignin fractions can be used as an internal indicator of thephenolic content of lignin wastes of different origin. Followingincubation for 24-48 hours with shaking at 30° C., the cultures can betested for growth upon exposure to the lignin fraction using an XTTassay kit. Culture samples can be removed from the 48 well growth plateand diluted appropriately in 96 well assay plates to which the XTTreagent can be added. The soluble formazan produced will be quantifiedby absorbance at 450 nm. Bacterial strains exhibiting the highest levelof growth, and therefore tolerance, can be candidates for furtherdevelopment as host strains for lignin conversions.

The strain demonstrated to have the best tolerance characteristics canbe transformed with the beta-etherase gene identified as showing thehighest biochemical activity. Restriction digestion can be used toexcise the ORF fragment from the cloning vector, and the sequence can besub-cloned into the shuttle vector pMMB206. Constructs cloned in theshuttle vector can be transformed into Azotobacter or Pseudomonasstrains by electroporation, or chemical transformation. The recombinant,lignin tolerant host strain can be re-tested for beta-etheraseexpression and activity using any methods known to one of skill, such asthose described herein, adapted to the particular host strain beingused.

Feedstock from Biorefinery Processes

An example of a starting material might be pretreated lignocellulosicbiomass. In some embodiments, the lignocellulose biomass material mightinclude grasses, corn stover, rice hull, agricultural residues,softwoods and hardwoods. In some embodiments, the lignin-derivedcompounds might be derived from hardwood species such as poplar from theUpper Peninsula region of Michigan, or hardwoods such as poplar, lollobypine, and eucalyptus from Virginia and Georgia areas, or mixed hardwoodsincluding maple and oak species from upstate New York.

In some embodiments, the pretreatment methods might encompass a range ofphysical, chemical and biological based processes. Examples ofpretreatment methods used to generate the feedstock for Aligna processesmight include physical pretreatment, solvent fractionation, chemicalpretreatment, biological pretreatment, ionic liquids pretreatment,supercritical fluids pretreatment, or a combination thereof, forexample, which can be applied in stages.

Physical pretreatment methods used to reduce the lignocellulose biomassparticle size reduction might utilize mechanical stress methods of dry,wet vibratory and compression-based ball milling procedures. Solventfractionation methods include organosolve processes, phosphoric acidfractionation processes, and methods using ionic liquids to pretreat thelignocellulose biomass to differentially solubilize and partitionvarious components of the biomass. In some embodiments, organosolvemethods might be performed using alcohol, including ethanol, with anacid catalyst at temperature ranges from about 90 to about 20° C., andfrom about 155 to about 220° C. with residence time of about 25 minutesto about 100 minutes. Catalyst concentrations can vary from about 0.83%to about 1.67% and alcohol concentrations can vary from about 25% toabout 74% (v/v). In some embodiments, phosphoric acid fractionations oflignocellulose biomass might be performed using a series of differentextractions using phosphoric acid, acetone, and water at temperature ofaround 50° C. In some embodiments, ionic liquid pretreatment oflignocellulose biomass might include use of ionic liquids containinganions like chloride, formate, acetate, or alkylphosphonate, withbiomass:ionic liquids ratios of approximately 1:10 (w/w). Thepretreatment might be performed at temperatures ranging from about 100°C. to about 150° C. Other ionic liquid compounds that might be usedinclude 1-butyl-3-methyl-imidazolium chloride and1-ethyl-3-methylimidazolium chloride.

Chemical pretreatments of lignocellulose biomass material might beperformed using technologies that include acidic, alkaline and oxidativetreatments. In some embodiments, acidic pretreatment methods oflignocellulose biomass such as those described below might be applied.Dilute acid pretreatments using sulfuric acid at concentrations in theapproximate range of about 0.05% to about 5%, and temperatures in therange of about 160° C. to about 220° C. Steam explosion, with or withoutthe use of catalysts such as sulfuric acid, nitric acid, carbonic acid,succinic acid, fumaric acid, maleic acid, citric acid, sulfur dioxide,sodium hydroxide, ammonia, before steam explosion, at temperaturesbetween about 160° C. to about 290° C. Liquid hot water treatment atpressure >5 MPa at temperatures ranging from about 160° C. to about 230°C., and pH range between about 4 and about 7. And, in some embodiments,alkaline pretreatment methods using catalysts such as calcium oxide,ammonia, and sodium hydroxide might be used. The ammonia fiber expansion(AFEX) method might be applied in which concentrated ammonia at about0.3 kg to about 2 kg of ammonia per kg of dry weight biomass is used atabout 60° C. to about 140° C. in a high pressure reactor, and cooked for5-45 minutes before rapid pressure release. The ammonia recyclepercolation (ARP) method might be used in flow through mode bypercolating ammoniacal solutions at 5-15% concentrations at hightemperatures and pressures. Oxidative pretreatment methods such asalkaline wet oxidation might be used with sodium carbonate at atemperature ranging from about 170° C. to about 220° C. in a highpressure reactor using pressurized air/oxygen mixtures or hydrogenperoxide as the oxidants.

Biological pretreatment methods using white rot basidomycetes andcertain actinomycetes might be applied. One type of product stream fromsuch pretreatment methods might be soluble lignin, and might containlignin-derived monomers and oligomers in the range of about 1 g/L toabout 10 g/L, and xylans. The lignin-derived monomers might includecompounds such as gallic acid, hydroxybenzoate, ferulic acid,hydroxymethyl furfural, hydroxymethyl furfural alcohol, vanillin,homovanillin, syringic acid, syringaldehyde, and furfural alcohol.

Supercritical fluid pretreatment methods might be used to process thebiomass. Examples of supercritical fluids for use in processing biomassinclude ethanol, acetone, water, and carbon dioxide at a temperature andpressures above the critical points for ethanol and carbon dioxide butat a temperature and/or pressure below that of the critical point forwater.

Combinations of steam pretreatment and biological pretreatment methodsmight be applied. For example, a biomass steam can be pretreated at 195°C. for 10 min at controlled pH, followed by enzymatic treatment usingcommercial cellulases and xylanases at dosings of 100 mg protein/g totalsolid, and with incubation at 50° C. at pH 5.0 with agitation of 500rpm.

In some embodiments, combinations of hydrothermal, organosolve, andbiological pretreatment methods might be used. One example of such acombination is a 3 stage process:

-   -   Stage 1. Use heat in an aqueous medium at a predetermined pH,        temperature and pressure for the hydrothermal process;    -   Stage 2. Use at least one organic solvent from those described        in 6-6c in water for the organosolve step;    -   Stage 3. Use yeast, white rot basidomycetes, actinomycetes, and        cellulases and xylanases in native or recombinant forms for the        biological pretreatment step.

Soluble lignin fractions derived using organosolve methods might producesoluble lignins in the molecular weight range of 188-1000, soluble invarious polar solvents. Without intending to be bound by any theory ormechanism of action, organosolve processes are generally believed tomaintain the lignin beta-aryl ether linkage.

Lignin streams from steam exploded lignocellulosic biomass might beused. Steam explosion might be performed, for example, using highpressure steam in the range of about 200 psi to about 500 psi, and attemperatures ranging from about 180° C. to about 230° C. for about 1minute to about 20 minutes in batch or continuous reactors. The ligninmight be extracted from the steam-exploded material with alkali washingor extraction using organic solvents. Steam exploded lignins can exhibitproperties similar to those described form organosolve lignins,retaining native bond structures and containing about 3 to about 12aromatic units per oligomer unit.

Supercritical fluid pretreatment can produce soluble lignin fractionsthat can be used with the teachings provided herein. Such processestypically yield monomers and lignin oligomers having a molecular weightof about <1000 Daltons.

Biological pretreatment can produce soluble lignin fractions that can beused with the teachings provided herein. Such lignin streams mightcontain lignin monomers and oligomers in the range of about 1 g/L toabout 10 g/L and have a molecular weight of about <1000 Daltons, andxylans. The lignin-derived monomers might include compounds such asgallic acid, hydroxybenzoate, ferulic acid, hydroxymethyl furfural,hydroxymethyl furfural alcohol, vanillin, homovanillin, syringic acid,syringaldehyde, and furfural alcohol.

Feedstock from Wood Pulping Processes

Wood pulping processes produce a variety of lignin types, the type oflignin dependent on the type of process used. Chemical pulping processesinclude, for example, Kraft and sulfite pulping.

In some embodiments, the lignin-derived compound can be derived from aspent pulping liquor or “black liquor” from Kraft pulping processes.Kraft lignin might be derived from batch or continuous processes using,for example, reaction temperatures in the range of about 150° C. toabout 200° C. and reaction times of approximately 2 hours. Any range ofmolecular weights of lignin may be obtained, and the useful fraction mayrange, in some embodiments, from about 200 Daltons to about 4000Daltons. A Kraft lignin having a molecular weight ranging from about1000 Daltons to about 3000 Daltons might be used in a bioconversion.

In some embodiments, lignin from a sulfite pulping process might beused. A sulfite pulping process can include, for example, a chemicalsulfonation using aqueous sulfur dioxide, bisulfite and monosulfite at apH ranging from about 2 to about 12. The sulfonated lignin might berecovered by precipitation with excess lime as lignosulfonates.Alternatively, formaldehyde-based methylation of the lignin aromaticsfollowed by sulfonation might be performed. Any range of molecularweights of lignin may be obtained, and the useful fraction may range, insome embodiments, from about 200 Daltons to about 4000 Daltons. Asulfite lignin having a molecular weight ranging from about 1000 Daltonsto about 3000 Daltons might be used in a bioconversion.

Characterization of Lignin-Derived Compounds for Use in Bioconversion

Optimization of a system for a particular feedstock should include anunderstanding of the composition of the particular feedstock. Forexample, one of skill will appreciate that the composition of a nativelignin can be significantly different than the composition of thelignin-derived compounds in a given lignin faction that is used for afeedstock. Accordingly, and understanding of the composition of thefeedstock will assist in optimizing the conversion of the lignin-derivedcompounds to the valuable aromatic compounds. Any method known to one ofskill can be used to characterize the compositions of the feedstock. Forexample, one of skill may use wet chemistry techniques, such asthioacidolysis and nitrobenzene oxidation, coupled with gaschromatography, which have been used traditionally, or spectroscopictechniques such as NMR and FTIR. Thioacidolysis, for example, cleavesthe β-O-4 linkages in lignin, giving rise to monomers and dimers whichare then used to calculate the S and G content. Similar information canbe obtained using nitrobenzene oxidation, but the ratios are thought tobe less accurate. In some embodiments, the content of S, G, and H, aswell as their relative ratios can be used to characterize feedstockcompositions for purposes of determining a bioconversion system design.

It is widely accepted that the biosynthesis of lignin stems from thepolymerization of three types of phenylpropane units, also referred toas monolignols. These units are coniferyl, sinapyl, and p-coumarylalcohol. The three structures are as follows:

Tables 1A and 1B summarize distributions of p-coumaryl alcohol orp-hydroxyl phenol (H), coniferyl alcohol or guaiacyl (G), and sinapylalcohol or syringyl (S) lignin in several sources of biomass. Table 1Acompares percent lignin in the biomass to the G:S:H.

TABLE 1A % Lignin G: S: H Wheat Straw 16-21 45 46 9 Rice Straw  6 45 4015 Rye Straw 18 43 53 1 Hemp  8-13 51 40 9 Tall Fescue: Stems  7-10 5542 3 Internodes 11 48 50 2 Flax 21-34 67 29 4 Jute 15-26 36 62 2 Sisal 7-14 22 76 2 Curaua Leaf fiber  7 29 41 30 Banana Plant Leaf 43 50 7Piassava Fiber 45 40 9 51 (Plam Tree) Abaca 7-9 19 55 26 Loblolly Pine29 86 2 12 29 87 0 13 Compression 60 40 Spruce (Picea Abies) 28 94 1 5MWL 98 2 0 Eucalyptus globus 22 14 84 2 Eucalyptus grandis 27 27 69 4Birch pendula 22 29 69 2 Beech 26 56 40 4 Acacia 28 48 49 3

Table 1A compares location of a sample in the biomass, species, andenvironmental stress to the G:S:H.

TABLE 1B White Birch G: S Fiber, S2 layer 12 88 Vessel, S2 Layer 88 12Ray parenchyma, S-layer 49 51 Middle lamella (fiber/fiber) 91 9 Middlelamella (fiber/vessel) 80 20 Middle lamella (fiber/ray) 100 0 Middlelamella (ray/ray) 88 12 G: S: H Lignin Samples 

Carpinus betulus MWL 19 80 1 Eucryphia codrifolia MWL 35 59 6 Bambusasp. MWL 23 57 20 Fagus sylvatica kraft lignin 25 72 3 Eucalyptusglobulus kraft lignin 22 73 6 Lobolly Pine Juvenile Normal 95 5 WindOpposite 96 4 Wind Compression 89 11 Bent Opposite 96 4 Bent Compression88 12

indicates data missing or illegible when filed

In general, the relative amounts of G, S, and H in lignin can be a goodindicator of its overall composition and response to a treatment, suchas the bioconversions taught herein. In poplar species, for example,differences can be seen based on the measurement technique as well asspecies, but in general the S/G ratio ranges from 1.3 to 2.2. This issimilar to the hardwood eucalyptus, but higher than herbaceous biomassswitchgrass and Miscanthus. This is to be expected given the higher Hcontents in grass lignin. An optimized nitrobenzene oxidation method hasshown S/G ratios of 13 poplar samples from two different sites andobtained values ranging from 1.01 to 1.68. Further, a linear correlation(R²=0.85) has been found in poplar between decreasing lignin content andincreasing S/G ratios. The correlation was stronger (R²=0.93) in samplesfrom a single site suggesting a dependency on geographic location.

Higher throughput methods can be used for rapid screening of feedstocks.Examples of such methods can include, but are not limited to,near-infrared (NIR), reflectance spectroscopy, pyrolysis molecular beammass spectrometry (pyMBMS), Fourier transform infrared spectroscopy, amodified thioacidolysis technique, and whole cell NMR after dissolutionin ionic liquids. Information on some structural characteristics oflignin, such as S/G ratios, can be rapidly obtained using these methods.The average S:G:H ratio of 104 poplar lignin samples, for example, wasdetermined using the modified thioacidolysis technique, and was found tobe 68:32:0.02. In some embodiments, the S, G, and H components in theratio can be expressed as mass percent. In some embodiments, the S, G,and H components in the ratio can be expressed as any relative unit, orunitless. Any comparison can be used, if the amount of each componentdirectly correlates with the other respective components in thecomposition. The ratios can be expressed in relative whole numbers orfractions as S:G:H, or any other order or combination of components,S/G, G/S, and the like. In some embodiments, the S/G ratio is used. Insome embodiments, the S/G ratio can range from about 0.20 to about 20.0,from about 0.3 to about 18.0, from about 0.4 to about 15.0, from about0.5 to about 15.0, from about 0.6 to about 12.0, from about 0.7 to about10.0, from about 0.8 to about 8.0, from about 0.9 to about 9.0, fromabout 1.0 to about 7.0, or any range therein. In some embodiments, theS/G ratio can be about 0.2, about 0.4, about 0.6, about 0.8, about 1.0,about 1.2, about 1.4, about 1.6, about 1.8, about 2.0, about 2.2, about2.4, about 2.6, about 2.8, about 3.0, about 3.2, about 3.4, about 3.6,about 3.8, about 4.0, about 4.2, about 4.4, about 4.6, about 4.8, about5.0, about 5.2, about 5.4, about 5.6, about 5.8, about 6.0, about 6.2,about 6.4, about 6.6, about 6.8, about 7.0, about 7.2, about 7.4, about7.6, about 7.8, about 8.0, about 8.2, about 8.4, about 8.6, about 8.8,about 9.0, about 9.2, about 9.4, about 9.6, about 9.8, about 10.0, andany ratio in-between on 0.1 increments, and any range of ratios therein.

Fractionation of Lignin-Derived Compounds for Use in Bioconversion

Soluble lignin streams derived from biorefinery or Kraft processes mightbe used directly in microbial conversions without additionalpurification or, they might be further purified by one or more of theseparation or fractionation techniques prior to microbial conversions.

In some embodiments, membrane filtration might be applied to achieve astarting concentration of lignin monomers and oligomers in the 1-60%(w/v) concentration range, and molecular weights ranging from about 180Daltons to about 2000 Daltons, from about 200 Daltons to about 4000Daltons, from about 250 Daltons to about 2500 Daltons, from about 180Daltons to about 3500 Daltons, from about 300 Daltons to about 3000Daltons, or any range therein.

In some embodiments, soluble lignin streams might be partially purifiedby chromatography using, for example, HP-20 resin. The lignin monomersand oligomers can bind to the resin while highly polar impurities orinorganics that might be toxic to microorganisms can remain un-bound.Subsequent elution, for example, with a methanol-water solvent system,can provide fractions of higher purity that are enriched in ligninmonomers and oligomers.

Chemical Products

A purpose of the present teaching includes the discovery of novelbiochemical conversions that create valuable commercial products fromvarious lignin core structures. Such commercial products includemonomeric aromatic chemicals that can serve as building block chemicals.One of skill will appreciate that a vast number of aromatic chemicalscan be produced using the principles provided by the teachings set-forthherein, and that a comprehensive teaching of every possible chemicalthat can be produced would be beyond the scope and purpose of thisteaching.

FIGS. 2A and 2B illustrate (i) the structures of some building blockchemicals that can be produced using bioconversions, and (ii) an exampleenzyme system from a Sphingomonaas paucimobilis gene cluster, accordingto some embodiments. FIG. 2A shows that examples of some monomericaromatic structures that can serve as building block chemicals derivedfrom lignin include, but are not limited to, guaiacol,β-hydroxypropiovanillone, 4-hydroxy-3 methoxy mandelic acid,coniferaldehyde, ferulic acid, eugenol, propylguaiacol, and4-acetylguaiacol. It should be appreciated that each of these structurescan be produced using the teachings provided herein. FIG. 2B(i) showsthe organization of the LigDFEG gene cluster in a Sphingomonaaspaucimobilis strain. FIG. 2B(ii) shows deduced functions of the geneproducts believed to be involved in a β-aryl ether bond cleavage in amodel lignin structure, guaiacylglycerol-β-guaiacyl ether (GGE). Thevertical bars above the restriction map indicate the positions of thegene insertions of LigD, LigF, LigE, and LigG. LigD shoedCα-dehydrogenase activity, LigF and LigE showed β-etherase activity, andLigG showed glutathione lyase activity. FIG. 2 LEGEND (Abbreviations):restriction enzymes Ap (ApaI), Bs (BstXI), E (EcoRI), Ec (Eco47III), Ml(MluI), P (PstI), RV (EcoRV), S, (SalI), Sc (SacI). ScII (SacII), St(StuI), Sm (SmaI), Tt (TthIIII), and X (XhoI); chemicals GGE(guaiacylglycerol-β-guaiacyl ether), GSH (glutathione), GSSG(glutathione disulfide), and asterisks are asymmetric carbons.

Commercial products that can be obtained from a bioconversion oflignin-derived compounds, as taught herein, include mono-aromaticchemicals. Examples of such chemicals include, but are not limited to,caprolactam, cumene, styrene, mononitro- and dinitrotoluenes and theirderivatives, 2,4-diaminotoluene, 2,4-dinitrotoluene, terephthalic acid,catechol, vanillin, salicylic acid, aminosalicylic acid, cresol andisomers, alkylphenols, chlorinated phenols, nitrophenols, polyhydricphenols, nitrobenzene, aniline and secondary and tertiary aniline bases,benzothiazole and derivatives, alkylbenzene and alkylbenzene sulfonates,4,4-diphenylmethane diisocyanate (MDI), chlorobenzenes anddichlorobenzenes, nitrochlorobenzenes, sulfonic acid derivatives oftoluene, pseudocumene, mesitylene, nitrocumene, cumenesulfonic acid.

Enzyme Discovery

The teachings herein are also directed to the discovery of novelenzymes. In some embodiments, the enzymes are beta-etherase enzymes.

Lignin is the only plant biomass constituent based on aromatic corestructures, and is comprised of branched phenylpropenyl (C9) units. Theguaiacol and syringol building blocks of lignin are linked throughcarbon-carbon (C—C) and carbon-oxygen (C—O, ether) bonds. The nativestructure of lignin suggests its key application as a chemical feedstockfor aromatic chemicals. The production of such chemical structuresnecessitates depolymerization and rupture of C—C and C—O bonds. Anabundant chemical linkage in lignin is the beta-aryl ether linkage,which comprises 50% to 70% of the bond type in lignin. The efficientscission of the beta-aryl ether bond would generate the monomericbuilding blocks of lignin, and provide the chemical feedstock forsubsequent conversion to a range of industrial products.

The beta-etherase enzyme system has multiple advantages for conversionsof lignin oligomers to monomers over the laccase enzyme systems. Thebeta-etherase enzyme system would achieve highly selective reductivebond scission catalysis for efficient and high yield conversions oflignin oligomers to monomers without the formation of side products,degradation of the aromatic core structures of lignin, or the use ofelectron transfer mediators required with use of the oxidative andradical chemistry-based laccase enzyme systems.

FIG. 3 is an example of a beta-etherase catalyzed hydrolysis of a modellignin dimer, α-O-(β-methylumbelliferyl) acetovanillone (MUAV),according to some embodiments. The scission of the beta-aryl ether bondin model compounds of lignin by beta-etherases from the microbeSphingmonas paucimobilis has been described. However, the availableinformation is limited, and there is no precedent in the literature forthe use of S. paucimobilis as an industrial microbe for commercial scaleprocesses. The discovery of new beta-etherase enzymes, and theheterologous expression of these new enzymes in Azotobacter strains willprovide the art with valuable industrial strains that particularlywell-suited for lignin conversion processes.

One of skill will recognize the chemical nomenclature used herein asstandard to the art. For example, the amino acids used herein can beidentified by at least the following conventional three-letterabbreviations in Table 2:

TABLE 2 Alanine A Ala Leucine L Leu Arginine R Arg Lysine K LysAsparagine N Asn Methionine M Met Aspartic acid D Asp Phenylalanine FPhe Cysteine C Cys Proline P Pro Glutamic acid E Glu Serine S SerGlutamine Q Gln Threonine T Thr Glycine G Gly Tryptophan W Trp HistidineH His Tyrosine Y Tyr Isoleucine I Ile Valine V Val Ornithine O Orn OtherXaa

The single letter identifier is provided for ease of reference, but anyformat can be used. The three-letter abbreviations are generallyaccepted in the peptide art, recommended by the IUPAC-IUB commission inbiochemical nomenclature, and are provided to comply with WIPO StandardST.25. Furthermore, the peptide sequences are taught according to thegenerally accepted convention of placing the N-terminus on the left andthe C-terminus on the right of the sequence listing to again comply withWIPO Standard ST.25.

The Recombinant Polypeptides

The teachings herein are based on discovery of novel and non-obviousproteins, DNAs, and host cell systems that can function in theconversion of lignin-derived compounds into valuable aromatic compounds.The systems can include natural, wild-type components or recombinantcomponents, the recombinant components being isolatable from what occursin nature.

The term “isolated” means altered “by the hand of man” from its naturalstate; i.e., if it occurs in nature, it has been changed or removed fromits original environment, or both. For example, a naturally occurringpolynucleotide or a polypeptide naturally present in a living animal inits natural state is not “isolated,” but the same polynucleotide orpolypeptide separated from the coexisting materials of its natural stateis “isolated”, as the term is used herein. For example, with respect topolynucleotides, the term isolated means that it is separated from thechromosome and cell in which it naturally occurs. However, a nucleicacid molecule contained in a clone that is a member of a mixed clonelibrary (e.g., a genomic or cDNA library) and that has not been isolatedfrom other clones of the library (e.g., in the form of a homogeneoussolution containing the clone without other members of the library) or achromosome isolated or removed from a cell or a cell lysate (e.g., a“chromosome spread”, as in a karyotype), is not “isolated” for thepurposes of the teachings herein. Moreover, a lone nucleic acid moleculecontained in a preparation of mechanically or enzymatically cleavedgenomic DNA, where the isolation of the nucleic molecule was not thegoal, is also not “isolated” for the purposes of the teachings herein.As part of, or following, an intentional isolation, polynucleotides canbe joined to other polynucleotides, for mutagenesis, to form fusionproteins, and for propagation or expression in a host, for instance.Isolated polynucleotides, alone or joined to other polynucleotides suchas vectors, can be introduced into host cells, in culture or in wholeorganisms, after which such DNAs still would be isolated, as the term isused herein, because they would not be in their naturally occurring formor environment. Similarly, the isolated polynucleotides and polypeptidesmay occur in a composition, such as a media formulation, solutions forintroduction of polynucleotides or polypeptides, for example, intocells, compositions or solutions for chemical or enzymatic reactions,for instance, which are not naturally occurring compositions, and,therein remain “isolated” polynucleotides or polypeptides within themeaning of that term as it is used herein.

A “vector,” such as an expression vector, is used to transfer ortransmit the DNA of interest into a prokaryotic or eukaryotic host cell,such as a bacteria, yeast, or a higher eukaryotic cell. Vectors can berecombinantly designed to contain a polynucleotide encoding a desiredpolypeptide. These vectors can include a tag, a cleavage site, or acombination of these elements to facilitate, for example, the process ofproducing, isolating, and purifying a polypeptide. The DNA of interestcan be inserted as the expression component of a vector. Examples ofvectors include plasmids, cosmids, viruses, and bacteriophages. If thevector is a virus or bacteriophage, the term vector can include theviral/bacteriophage coat. The term “expression vector” is usually usedto describe a DNA construct containing gene encoding an expressionproduct of interest, usually a protein, that is expressed by themachinery of the host cell. This type of vector is frequently a plasmid,but the other forms of expression vectors, such as bacteriophage vectorsand viral vectors (e.g., adenoviruses, replication defectiveretroviruses, and adeno-associated viruses), can be used.

In some embodiments, the polypeptides taught herein can be natural orwildtype, isolated and/or recombinant. In some embodiments, thepolynucleotides can be natural or wildtype, isolated and/or recombinant.In some embodiments, the teachings are directed to a vector than caninclude such a polynucleotide or a host cell transformed by such avector.

In some embodiments, the polypeptide can be an isolated recombinantpolypeptide, comprising an amino acid sequence having at least 95%identity to SEQ ID NO:101. The sequence can conserve residues T19, I20,S21, P22, V24, W25, T27, K28, Y29, A30, H33, K34, G35, F36, D39, I40,V41, P42, G43, G44, F45, G47, I48, E50, R51, T52, G53, G54, K100, A101,N104, V111, G112, M115, F116, P166, W107, Y184, Y187, R188, G191, G192,and F195.

In some embodiments, the polypeptide can be an isolated recombinantpolypeptide, comprising SEQ ID NO:101; or conservative substitutionsthereof outside of the conserved residues. The conserved residues caninclude T19, I20, S21, P22, V24, W25, T27, K28, Y29, A30, H33, K34, G35,F36, D39, I40, V41, P42, G43, G44, F45, G47, I48, E50, R51, T52, G53,G54; K100, A101, N104, V111, G112, M115, F116, P166, W107, Y184, Y187,R188, G191, G192, and F195.

In some embodiments, the polypeptide can be an isolated recombinantglutathione S-transferase enzyme, comprising an amino acid sequencehaving at least 95% identity to SEQ ID NO:101. The amino acid sequencecan conserve residues T19, I20, S21, P22, V24, W25, T27, K28, Y29, A30,H33, K34, G35, F36, D39, I40, V41, P42, G43, G44, F45, G47, I48, E50,R51, T52, G53, G54; K100, A101, N104, V111, G112, M115, F116, P166,W107, Y184, Y187, R188, G191, G192, and F195; wherein, the amino acidsequence functions to cleave a beta-aryl ether.

In some embodiments, the polypeptide can be an isolated recombinantglutathione S-transferase enzyme, comprising an amino acid sequencehaving at least 95% identity to SEQ ID NO:101; wherein, the amino acidsequence functions to cleave a beta-aryl ether.

In some embodiments, the polypeptide can be an isolated recombinantpolypeptide, comprising (i) a length ranging from about 279 to about 281amino acids; (ii) a first amino acid region consisting of residues 19-54from SEQ ID NO:101, or conservative substitutions thereof outside ofconserved residues T19, I20, S21, P22, V24, W25, T27, K28, Y29, A30,H33, K34, G35, F36, D39, I40, V41, P42, G43, G44, F45, G47, I48, E50,R51, T52, G53, and G54; wherein, the first amino acid region can belocated in the recombinant polypeptide from about residue 14 to aboutresidue 59; and, (iii) a second amino acid region consisting of residues98-221 from SEQ ID NO:101, or conservative substitutions thereof outsideof conserved residues K100, A101, N104, V111, G112, M115, F116, P166,W107, Y184, Y187, R188, G191, G192, and F195; wherein, the second aminoacid region is located in the recombinant polypeptide from about residue93 to about residue 226.

In some embodiments, the polypeptide can be an isolated recombinantglutathione S-transferase enzyme, comprising (i) a length ranging fromabout 279 to about 281 amino acids; (ii) a first amino acid regionhaving at least 95% identity to residues 19-54 from SEQ ID NO:101 whileconserving residues T19, I20, S21, P22, V24, W25, T27, K28, Y29, A30,H33, K34, G35, F36, D39, I40, V41, P42, G43, G44, F45, G47, I48, E50,R51, T52, G53, and G54; wherein, the first amino acid region is locatedin the recombinant polypeptide from about residue 14 to about residue59; and, (iii) a second amino acid region having at least 95% identityto residues 98-221 from SEQ ID NO:101 while conserving residues K100,A101, N104, V111, G112, M115, F116, P166, W107, Y184, Y187, R188, G191,G192, and F195; wherein, the second amino acid region can be located inthe recombinant polypeptide from about residue 93 to about residue 226;and, the recombinant glutathione S-transferase enzyme can function tocleave a beta-aryl ether.

In some embodiments, the polypeptide can be an isolated recombinantglutathione S-transferase enzyme, comprising an amino acid sequencehaving at least 95% identity to SEQ ID NO:541; wherein, the amino acidsequence functions to cleave a beta-aryl ether.

In some embodiments, the polypeptide can be an isolated recombinantpolypeptide, comprising (i) a length ranging from about 256 to about 260amino acids; (ii) a first amino acid region consisting of residues 47-57from SEQ ID NO:541, or conservative substitutions thereof outside ofconserved residues A47, I48, N49, P50, G52, V54, P55, V56, L57; wherein,the first amino acid region is located in the recombinant polypeptidefrom about residue 45 to about residue 57; (iii) a second amino acidregion consisting of 63-76 from SEQ ID NO:541; and, (iv) a third aminoacid region consisting of residues 99-230 from SEQ ID NO:541, orconservative substitutions thereof outside of conserved residues R100,Y101, K104, D107, M111, N112, S115, M116, K176, L194, I197, N198, S201,H202, and M206; wherein, the second amino acid region is located in therecombinant polypeptide from about residue 94 to about residue 235.

In some embodiments, the polypeptide can be an isolated recombinantglutathione S-transferase enzyme, comprising (i) a length ranging fromabout 279 to about 281 amino acids; (ii) a first amino acid regionhaving at least 95% identity to 47-57 from SEQ ID NO:541, orconservative substitutions thereof outside of conserved residues A47,I48, N49, P50, G52, V54, P55, V56, L57; wherein, the first amino acidregion can be located in the recombinant polypeptide from about residue45 to about residue 57; (iii) a second amino acid region consisting of63-76 from SEQ ID NO:541; and, (iv) a third amino acid region having atleast 95% identity to residues 99-230 from SEQ ID NO:541, orconservative substitutions thereof outside of conserved residues R100,Y101, K104, D107, M111, N112, S115, M116, K176, L194, I197, N198, S201,H202, and M206; wherein, the second amino acid region can be located inthe recombinant polypeptide from about residue 94 to about residue 235;wherein, the recombinant glutathione S-transferase enzyme functions tocleave a beta-aryl ether.

In some embodiments, an amino acid substitution outside of the conservedresidues can be a conservative substitution. And, in many embodiments,the amino acid sequence can function to cleave a beta-aryl ether.

Methods of Preparing the Recombinant SDF-1 Polynucleotide andPolypeptides

The teachings include a method of preparing the polypeptides describedherein, comprising culturing a host cell under conditions suitable toproduce the desired polypeptide; and recovering the polypeptide from thehost cell culture; wherein, the host cell comprises anexogenously-derived polynucleotide encoding the desired polypeptide. Insome embodiments, the host cell is E. Coli. In some embodiments, thehost cell can be an Azotobacter strain such as, for example, Azotobactervinelandii.

Initially, a double-stranded DNA fragment encoding the primary aminoacid sequence of recombinant polypeptide can be designed. This DNAfragment can be manipulated to facilitate synthesis, cloning, expressionor biochemical manipulation of the expression products. The syntheticgene can be ligated to a suitable cloning vector and then the nucleotidesequence of the cloned gene can be determined and confirmed. The genecan be then amplified using designed primers having specific restrictionenzyme sequences introduced at both sides of insert gene, and the genecan be subcloned into a suitable subclone/expression vector. Theexpression vector bearing the synthetic gene for the mutant can beinserted into a suitable expression host. Thereafter the expression hostcan be maintained under conditions suitable for production of the geneproduct and, in some embodiments, the protein can be (i) isolated andpurified from the cells expressing the gene or (ii) used directly in areaction environment that includes the host cell.

The nucleic acid (e.g., cDNA or genomic DNA) may be inserted into areplicable vector for cloning (amplification of the DNA) for expression.Various vectors are publicly available. In general, DNA can be insertedinto an appropriate restriction endonuclease site(s) using techniquesknown in the art, for example. Vector components generally include, butare not limited to, one or more of a signal sequence, an origin ofreplication, one or more marker genes, an enhancer element, a promoter,and a transcription termination sequence.

The signal sequence may be a prokaryotic signal sequence selected, forexample, from the group of the alkaline phosphatase, penicillinase, Ipp,or heat-stable enterotoxin II leaders. For yeast secretion the signalsequence may be, e.g., the yeast invertase leader, alpha factor leader(including Saccharomyces and Kluyveromyces alpha-factor leaders, thelatter described in U.S. Pat. No. 5,010,182), or acid phosphataseleader, the C. albicans glucoamylase leader (EP 362,179), or the signaldescribed in WO 90/13646, for example. In mammalian cell expression,mammalian signal sequences may be used to direct secretion of theprotein, such as signal sequences from secreted polypeptides of the sameor related species, as well as viral secretory leaders.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells. Suchsequences are well known for a variety of bacteria, yeast, and viruses.The origin of replication from a plasmid, e.g. pBR322, for example, issuitable for most Gram-negative bacteria, and the 2μ plasmid origin issuitable for yeast, and various viral origins (SV40, polyoma,adenovirus, VSV or BPV) are useful for cloning vectors in mammaliancells.

Expression and cloning vectors will typically contain a selection gene,also termed a selectable marker. Typical selection genes encode proteinsthat (a) confer resistance to antibiotics or other toxins, e.g.,ampicillin, neomycin, methotrexate, or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g., the gene encoding D-alanine racemase forBacilli.

An example of suitable selectable markers for mammalian cells are thosethat enable the identification of cells competent to take the encodingnucleic acid, such as DHFR or thymidine kinase. An appropriate host cellwhen wild-type DHFR is employed is the CHO cell line deficient in DHFRactivity, prepared and propagated as described by Urlaub et al., Proc.Natl. Acad. Sci. USA, 77:4216 (1980). A suitable selection gene for usein yeast is the trp1 gene present in the yeast plasmid YRp7 (Stinchcombet al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979);Tschemper et al., Gene, 10:157 (1980)). The trp1 gene provides aselection marker for a mutant strain of yeast lacking the ability togrow in tryptophan, for example, ATCC No. 44076 or PEP4-1 (Jones,Genetics, 85:12 (1977)).

Expression and cloning vectors usually contain a promoter operablylinked to the encoding nucleic acid sequence to direct mRNA synthesis.Promoters recognized by a variety of potential host cells are wellknown. Promoters suitable for use with prokaryotic hosts include the.beta.-lactamase and lactose promoter systems (Chang et al., Nature,275:615 (1978); Goeddel et al., Nature, 281:544 (1979)), alkalinephosphatase, a tryptophan (trp) promoter system (Goeddel, Nucleic AcidsRes., 8:4057 (1980); EP 36,776), and hybrid promoters such as the tacpromoter (deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21 25 (1983)).Promoters for use in bacterial systems also will contain aShine-Dalgarno sequence operably linked to the encoding DNA.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are known in the art,e.g. see EP 73,657 for a further discussion.

PRO87299 transcription from vectors in mammalian host cells iscontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus (UK 2,211,504), adenovirus(such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40(SV40), from heterologous mammalian promoters, e.g., the actin promoteror an immunoglobulin promoter, and from heat-shock promoters, providedsuch promoters are compatible with the host cell systems.

Transcription of the encoding DNA by higher eukaryotes may be increasedby inserting an enhancer sequence into the vector. Enhancers arecis-acting elements of DNA, usually about from 10 to 300 bp, that act ona promoter to increase its transcription. Many enhancer sequences arenow known from mammalian genes (globin, elastase, albumin,a-fetoprotein, and insulin). Typically, however, one will use anenhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin, the cytomegalovirusearly promoter enhancer, the polyoma enhancer on the late side of thereplication origin, and adenovirus enhancers. The enhancer may bespliced into the vector at a position 5′ or 3′ to the coding sequencebut is preferably located at a site 5′ from the promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding the mutants.

In some embodiments, the expression control sequence can be selectedfrom a group consisting of a lac system, T7 expression system, majoroperator and promoter regions of pBR322 origin, and other prokaryoticcontrol regions. Still other methods, vectors, and host cells suitablefor adaptation to the synthesis of the mutants in recombinant vertebratecell culture are described in Gething et al., Nature, 293:620 625(1981); Mantei et al., Nature, 281:40 46 (1979); EP 117,060; and EP117,058.

Mutants can be expressed as a fusion protein. In some embodiments, themethods involve adding a number of amino acids to the protein, and insome embodiments, to the amino terminus of the protein. Extra aminoacids can serve as affinity tags or cleavage sites, for example. Fusionproteins can be designed to: (1) assist in purification by acting as atemporary ligand for affinity purification, (2) produce a preciserecombinant by removing extra amino acids using a cleavage site betweenthe target gene and affinity tag, (3) increase the solubility of theproduct, and/or (4) increase expression of the product. A proteolyticcleavage site can be included at the junction of the fusion region andthe protein of interest to enable further purification of theproduct—separation of the recombinant protein from the fusion proteinfollowing affinity purification of the fusion protein. Such enzymes, andtheir cognate recognition sequences, can include Factor Xa, thrombin andenterokinase, cyanogen bromide, trypsin, or chymotrypsin, for example.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S. Gene 67:31-40 (1988)), pMAL (New EnglandBiolabs, Beverly, Mass.), pRIT5 (Pharmacia, Piscataway, N.J.), and pET(Strategen), which can fuse glutathione S-transferase (GST), maltose Ebinding protein, protein A, or a six-histidine sequence, respectively,to a target recombinant protein.

Synthetic DNAs containing the sequences of nucleotides, tags andcleavage sites can be designed and provided as a modified coding forrecombinant polypeptide mutants. In some embodiments, a polypeptide canbe a fusion polypeptide having an affinity tag, and the recovering stepincludes (1) capturing and purifying the fusion polypeptide, and (2)removing the affinity tag for high yield production of the desiredpolypeptide or an amino acid sequence that is at least 95% homologous toa desired polypeptide. DNA encoding the mutants may be obtained from acDNA library prepared from tissue possessing the mRNA for the mutants.As such, the DNA can be conveniently obtained from a cDNA library. Theencoding gene for the mutants may also be obtained from a genomiclibrary or by known synthetic procedures (e.g., automated nucleic acidsynthesis).

Libraries can be screened with probes designed to identify the gene ofinterest or the protein encoded by it. Screening the cDNA or genomiclibrary with the selected probe may be conducted using standardhybridization procedures, such as described in Sambrook et al.,Molecular Cloning: A Laboratory Manual (New York: Cold Spring HarborLaboratory Press, 1989), which is herein incorporated by reference. Analternative means to isolate the gene encoding recombinant polypeptidemutants is to use PCR methodology [Sambrook et al., supra; Dieffenbachet al., PCR Primer: A Laboratory Manual (Cold Spring Harbor LaboratoryPress, 1995)].

Nucleic acids having a desired protein coding sequence may be obtainedby screening selected cDNA or genomic libraries using a deduced aminoacid sequence and, if necessary, a conventional primer extensionprocedure as described in Sambrook et al., supra, to detect precursorsand processing intermediates of mRNA that may not have beenreverse-transcribed into cDNA.

The selection of expression vectors, control sequences, transformationmethods, and the like, are dependent on the type of host cell used toexpress the gene. Following entry into a cell, all or part of the vectorDNA, including the insert DNA, may be incorporated into the host cellchromosome, or the vector may be maintained extrachromosomally. Thosevectors that are maintained extrachromosomally are frequently capable ofautonomous replication in the host cell. Other vectors are integratedinto the genome of a host cell upon and are replicated along with thehost genome.

Host cells are transfected or transformed with the expression or cloningvectors described herein to produce the mutants. The cells are culturedin conventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences. The culture conditions, such as media, temperature,pH and the like, can be selected by the skilled artisan without undueexperimentation. In general, principles, protocols, and practicaltechniques for maximizing the productivity of cell cultures can be foundin Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed.(IRL Press, 1991) and Sambrook et al., supra, each of which areincorporated by reference.

The host cells can be prokaryotic or eukaryotic and, suitable host cellsfor cloning or expressing the DNA in the vectors herein can includeprokaryote, yeast, or higher eukaryote cells. Methods of eukaryotic celltransfection and prokaryotic cell transformation are known to theordinarily skilled artisan, for example, CaCl2, CaPO4, liposome-mediatedand electroporation. Depending on the host cell used, transformation isperformed using standard techniques appropriate to such cells. Thecalcium treatment employing calcium chloride, as described in Sambrooket al., supra, or electroporation is generally used for prokaryotes.Infection with Agrobacterium tumefaciens is used for transformation ofcertain plant cells, as described by Shaw et al., Gene, 23:315 (1983)and WO 89/05859 published 29 Jun. 1989. For mammalian cells without suchcell walls, the calcium phosphate precipitation method of Graham and vander Eb, Virology, 52:456 457 (1978) can be employed. General aspects ofmammalian cell host system transfections have been described in U.S.Pat. No. 4,399,216. Transformations into yeast are typically carried outaccording to the method of Van Solingen et al., J. Bact., 130:946 (1977)and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However,other methods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene, polyornithine, may also be used.For various techniques for transforming mammalian cells, see Keown etal., Methods in Enzymology, 185:527 537 (1990) and Mansour et al.,Nature, 336:348 352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes include, but are not limited to, eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC53,635). Other suitable prokaryotic host cells includeEnterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimunrium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. These examples are illustrative ratherthan limiting, and merely supplement the remainder of the teachingsherein. Strain W3110 is one particularly preferred host or parent hostbecause it is a common host strain for recombinant DNA productfermentations. Preferably, the host cell secretes minimal amounts ofproteolytic enzymes. For example, strain W3110 may be modified to effecta genetic mutation in the genes encoding proteins endogenous to thehost, with examples of such hosts including E. coli W3110 strain 1 A2,which has the complete genotype tonA; E. coli W3110 strain 9E4, whichhas the complete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC55,244), which has the complete genotype tonA ptr3 phoA E15(argF-lac)169 degP ompT kanr; E. coli W3110 strain 37D6, which has thecomplete genotype tonA ptr3 phoA E15 (argF-lac) 169 degP ompT rbs7 ilvCkanr; E. coli W3110 strain 40B4, which is 37D6 with a non-kanamycinresistant degP deletion mutation; and an E. coli strain having mutantperiplasmic protease as disclosed in U.S. Pat. No. 4,946,783.Alternatively, in vitro methods of cloning, e.g., PCR or other nucleicacid polymerase reactions, are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for the mutants.Saccharomyces cerevisiae is a commonly used lower eukaryotic hostmicroorganism. Others include Schizosaccharomyces pombe (Beach andNurse, Nature, 290: 140 (1981); EP 139,383 published 2 May 1985);Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al.,Bio/Technology, 9:968 975 (1991)) such as, e.g., K. lactis (MW98-8C,CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 154(2):737 742(1983)), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K.wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum(ATCC 36,906; Van den Berg et al., Bio/Technology, 8:135 (1990)), K.thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris(EP 183,070; Sreekrishna et al., J. Basic Microbiol., 28:265 278[1988]); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa(Case et al., Proc. Natl. Acad. Sci. USA, 76:5259 5263 (1979));Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538); andfilamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium(WO 91/00357), and Aspergillus hosts such as A. nidulans (Ballance etal., Biochem. Biophys. Res. Commun., 112:284 289 (1983); Tilburn et al.,Gene, 26:205 221 (1983); Yelton et al., Proc. Natl. Acad. Sci. USA, 81:1470 1474 (1984)) and A. niger (Kelly and Hynes, EMBO J., 4:475 479(1985)) Methylotropic yeasts are suitable herein and include, but arenot limited to, yeast capable of growth on methanol selected from thegenera consisting of Hansenula, Candida, Kloeckera, Pichia,Saccharomyces, Torulopsis, and Rhodotorula. A list of specific speciesthat are exemplary of this class of yeasts may be found in C. Anthony,The Biochemistry of Methylotrophs, 269 (1982).

Suitable host cells for the expression of glycosylated mutants can bederived from multicellular organisms. Invertebrate cells include insectcells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells.Useful mammalian host cell lines include Chinese hamster ovary (CHO) andCOS cells. More specific examples include monkey kidney CVI linetransformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line(293 or 293 cells subcloned for growth in suspension culture, Graham etal., J. Gen Virol., 36:59 (1977)); Chinese hamster ovarycells/−DHFR(CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243 251(1980)); human lung cells (W138, ATCC CCL 75); human liver cells (HepG2, HB 8065); and mouse mammary tumor (MMT 060562, ATCC CCL5 1). One ofskill can readily choose the appropriate host cell, at least forextracellular protein harvesting embodiments, without undueexperimentation.

In some embodiments, a nucleotide sequence will be hybridizable, undermoderately stringent conditions, to a nucleic acid having a nucleotidesequence comprising or complementary to the desired nucleotidesequences. In some embodiments, an isolated nucleotide sequence will behybridizable, under stringent conditions, to a nucleic acid having anucleotide sequence comprising or complementary to the desirednucleotide sequences. A nucleic acid molecule can be “hybridizable” toanother nucleic acid molecule when a single-stranded form of the nucleicacid molecule can anneal to the other nucleic acid molecule under theappropriate conditions of temperature and ionic strength (see Sambrooket al., supra,). The conditions of temperature and ionic strengthdetermine the “stringency” of the hybridization. “Hybridization”requires that two nucleic acids contain complementary sequences.However, depending on the stringency of the hybridization, mismatchesbetween bases may occur. The appropriate stringency for hybridizingnucleic acids depends on the length of the nucleic acids and the degreeof complementation. Such variables are well known in the art. Morespecifically, the greater the degree of similarity or homology betweentwo nucleotide sequences, the greater the value of Tm for hybrids ofnucleic acids having those sequences. For hybrids of greater than 100nucleotides in length, equations for calculating Tm have been derived(see Sambrook et al., supra). For hybridization with shorter nucleicacids, the position of mismatches becomes more important, and the lengthof the oligonucleotide determines its specificity (see Sambrook et al.,supra).

In some embodiments, the polynucleotides and polypeptides have at least55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99percent homology to a desired polynucleotide or polypeptide. In someembodiments, the polynucleotides and polypeptides have at least 55, 60,65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percentidentity to a desired polynucleotide or polypeptide. And, in someembodiments, the polynucleotides and polypeptides have at least 55, 60,65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percentsimilarity to a desired polynucleotide or polypeptide. As describedabove, degenerate forms of the desired polynucleotide are alsoacceptable. In some embodiments, a polypeptide can be 90, 91, 92, 93,94, 95, 96, 97, 98, or 99 homologous, identical, or similar to a desiredpolypeptide as long as it shares the same function as the desiredpolypeptide, and the extent of the function can be less or more thanthat of the desired polypeptide. In some embodiments, for example, apolypeptide can have a function that is 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 100%, or any 0.1% increment in-between, that of thedesired polypeptide. And, in some embodiments, for example, apolypeptide can have a function that is 110%, 120%, 130%, 140%, 150%,160%, 170%, 180%, 190%, 200%, 300%, 400%, 500%, or more, or any 1%increment in-between, that of the desired polypeptide. In someembodiments the “function” is an enzymatic activity, measurable by anymethod known to one of skill such as, for example, a method used in theteachings herein. The “desired polypeptide” or “desired polynucleotide”can be referred to as a “reference polypeptide” or “referencepolynucleotide”, or the like, in some embodiments as a control forcomparison of a polypeptide of interest, which may be considered a “testpolypeptide” or “test polynucleotide” or the like. In any event, thecomparison is that of one set of bases or amino acids against anotherset for purposes of measuring homology, identity, or similarity. Theability to hybridize is, of course, another way of comparing nucleotidesequences.

The terms “homology” and “homologous” can be used interchangeably insome embodiments. The terms can refer to nucleic acid sequence matchingand the degree to which changes in the nucleotide bases betweenpolynucleotide sequences affects the gene expression. These terms alsorefer to modifications, such as deletion or insertion of one or morenucleotides, and the effects of those modifications on the functionalproperties of the resulting polynucleotide relative to the unmodifiedpolynucleotide. Likewise the terms refer to polypeptide sequencematching and the degree to which changes in the polypeptide sequences,such as those seen when comparing the modified polypeptides to theunmodified polypeptide, affect the function of the polypeptide. Itshould appreciated to one of skill that the polypeptides, such as themutants taught herein, can be produced from two non-homologouspolynucleotide sequences within the limits of degeneracy.

The terms “similarity” and “identity” are known in the art. The term“identity” can be used to refer to a sequence comparison based onidentical matches between correspondingly identical positions in thesequences being compared. The term “similarity” can be used to refer toa comparison between amino acid sequences, and takes into account notonly identical amino acids in corresponding positions, but alsofunctionally similar amino acids in corresponding positions. Thussimilarity between polypeptide sequences indicates functionalsimilarity, in addition to sequence similarity. Levels of identitybetween gene sequences and levels of identity or similarity betweenamino acid sequences can be calculated using known methods. For example,publicly available computer based methods for determining identity andsimilarity include the BLASTP, BLASTN and FASTA (Atschul et al., J.Molec. Biol., 1990; 215:403-410), the BLASTX program available fromNCBI, and the Gap program from Genetics Computer Group, Madison Wis. Insome embodiments, the Gap program, with a Gap penalty of 12 and a Gaplength penalty of 4 can be used for determining the amino acid sequencecomparisons, and a Gap penalty of 50 and a Gap length penalty of 3 forthe polynucleotide sequence comparisons. In some embodiments, thesequences can be aligned so that the highest order match is obtained.The match can be calculated using published techniques that include, forexample, Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991, each of which is incorporated by reference herein.

As such, the term “similarity” is similar to “identity”, but in contrastto identity, similarity can be used to refer to both identical matchesand conservative substitution matches. For example, if two polypeptidesequences have 10/20 identical amino acids, and the remainder are allnon-conservative substitutions, then the percent identity and similaritywould both be 50%. On the other hand, if there are 5 five more positionswhere there are conservative substitutions, then the percent identity is50%, whereas the percent similarity is 75%.

In some embodiments, the term “substantial sequence identity” can referto an optimal alignment, such as by the programs GAP or BESTFIT usingdefault gap penalties, having at least 65, 70, 75, 80, 85, 90, 95, 96,97, 98, or 99 percent sequence identity. The difference in what is“substantial” regarding identity can often vary according to acorresponding percent similarity, since the factor of primary importanceis often the function of the sequence in a system. The term “substantialpercent identity” can be used to refer to a DNA sequence that issufficiently similar to a reference sequence at the nucleotide level tocode for the same protein, or a protein having substantially the samefunction, in which the comparison can allow for allelic differences inthe coding region. Likewise, the term can be used to refer to acomparison of sequences of two polypeptides optimally aligned.

In some embodiments, sequence comparisons can be made to a referencesequence over a “comparison window” of amino acids or bases thatincludes any number of amino acids or bases that is useful in theparticular comparison. For example, the reference sequence may be asubset of a larger sequence. In some embodiments, the comparison windowcan include at least 10 residue or base positions, and sometimes atleast 15-20 amino acids or bases. The reference or test sequence mayrepresent, for example, a polypeptide or polynucleotide having one ormore deletions, substitutions or additions.

The term “variant” refers to modifications to a peptide that allows thepeptide to retain its binding properties, and such modificationsinclude, but are not limited to, conservative substitutions in which oneor more amino acids are substituted for other amino acids; deletion oraddition of amino acids that have minimal influence on the bindingproperties or secondary structure; conjugation of a linker;post-translational modifications such as, for example, the addition offunctional groups. Examples of such post-translational modifications caninclude, but are not limited to, the addition of modifying groupsdescribed below through processes such as, for example, glycosylation,acetylation, phosphorylation, modifications with fatty acids, formationof disulfide bonds between peptides, biotinylation, PEGylation, andcombinations thereof. In fact, in most embodiments, the polypeptides canbe modified with any of the various modifying groups known to one ofskill.

The terms “conservatively modified variant,” “conservatively modifiedsubstitution,” and “conservative substitution” can be usedinterchangeably in some embodiments. These terms can be used to refer toa conservative amino acid substitution, which is an amino acidsubstituted by an amino acid of similar charge density,hydrophilicity/hydrophobicity, size, and/or configuration such as, forexample, substituting valine for isoleucine. In comparison, a“non-conservatively modified variant” refers to a non-conservative aminoacid substitution, which is an amino acid substituted by an amino acidof differing charge density, hydrophilicity/hydrophobicity, size, and/orconfiguration such as, for example, substituting valine forphenyalanine. One of skill will appreciate that there are a plurality ofways to define conservative substitutions, and any of these methods maybe used with the teachings provided herein. In some embodiments, forexample, a substitution can be considered conservative if an amino acidfalling into one of the following groups is substituted by an amino acidfalling in the same group: hydrophilic (Ala, Pro, Gly, Glu, Asp, Gln,Asn, Ser, Thr), aliphatic (Val, Ile, Leu, Met), basic (Lys, Arg, H is),aromatic (Phe, Tyr, Trp), and sulphydryl (Cys). See Dayhoff, M O. Et al.National Biomedical Research Foundation, Georgetown University,Washington D.C.:89-99 (1972), which is incorporated herein. In someembodiments, the substitution of amino acids can be consideredconservative where the side chain of the substitution has similarbiochemical properties to the side chain of the substituted amino acid.

Microbial Systems—Antimicrobial Lignin-Derived Compounds

The antimicrobial activity of lignin-derived compounds is a majorproblem addressed by the systems taught herein. For example, typicalindustrial fermentation processes might utilize the microbes Escherichiacoli K12 or Escherichia coli B, or the yeast Saccharomyces cerevisiae,and recombinant versions of these microbes, which are well characterizedindustrial strains. The problem is that the antimicrobial activities ofaromatic compounds on such industrial microbes are toxic to themicrobes, which negates an application to biotransformations oflignin-derived compounds.

The phenolic streams or soluble lignin streams derived from pretreatedlignocellulosic biomass, for example, might contain aromatic andnonaromatic compounds, such as gallic acid, hydroxymethylfurfuralalcohol, hydroxymethylfurfural, furfural alcohol, 3,5-dihydroxybenzoate,furoic acid, 3,4-dihydroxybenzaldehyde, hydroxybenzoate, homovanillin,syringic acid, vanillin, and syringaldehyde. There are severallignin-derived compounds that are antimicrobials. For example, furfural,4-hydroxybenzaldehyde, syringaldehyde, 5-hydroxymethylfurfural, andvanillin are each known to have antimicrobial activity againstEscherichia coli, and might have an additive antimicrobial activityagainst Escherichia coli when present in combination. Moreover,veratraldehyde, cinnamic acid and the respective benzoic acidderivatives of vanillic acid, vanillylacetone, and the cinnamic acidderivatives o-coumaric acid, m-coumaric acid, and p-coumaric acid mightbe components of the phenolic streams from pretreated lignocellulosicbiomass. Veratraldehyde, cinnamic acid and the respective benzoic acidderivatives of vanillic acid, vanillylacetone, and cinnamic acidderivatives o-coumaric acid, m-coumaric acid, and p-coumaric acid, eachhave significant antifungal activities against the yeast Saccharomycescerevisiae, and might have an additive antifungal activity against theyeast Saccharomyces cerevisiae when present in combination.

One or more of the following benzaldehyde derivatives might be presentin the phenolic streams from pretreated lignocellulosic biomass:2,4,6-trihydroxybenzaldehyde, 2,5-dihydroxybenzaldehyde,2,3,4-trihydroxybenzaldehyde, 2-hydroxy-5-methoxybenzaldehyde,2,3-dihydroxybenzaldehyde, 2-hydroxy-3-methoxybenzaldehyde,4-hydroxy-2,6-dimethoxybenzaldehyde, 2,5-dihydroxybenzaldehyde,2,4-dihydroxybenzaldehyde, and 2-hydroxybenzaldehyde. Likewise,2,4,6-trihydroxybenzaldehyde, 2,5-dihydroxybenzaldehyde,2,3,4-trihydroxybenzaldehyde, 2-hydroxy-5-methoxybenzaldehyde,2,3-dihydroxybenzaldehyde, 2-hydroxy-3-methoxybenzaldehyde,4-hydroxy-2,6-dimethoxybenzaldehyde, 2,5-dihydroxybenzaldehyde,2,4-dihydroxybenzaldehyde, and 2-hydroxybenzaldehyde have eachdemonstrated antibacterial activity against Escherichia coli, and mighthave an additive antibacterial activity against Escherichia coli whenpresent in combination.

Microbial Systems—Suitable Microbes

The antimicrobial activity of lignin-derived compounds creates a needfor a strain of microbe that is tolerant to such activity in thereaction environment. The teachings include the identification ofrecombinant or non-recombinant microbial species that are naturallycapable of metabolizing aromatic compounds for the biotransformations oflignin-derived compounds to commercial products.

Some examples of microbial species particularly suited forbiotransformations of phenolic streams from pretreated lignocellulosicbiomass include, but are not limited to, Azotobacter chroococcum,Azotobacter vinelandii, Novosphingobium aromaticivorans, Pseudomonasaeruginosa, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonasstutzerii, Pseudomonas diminuta, Pseudomonas pseudoalcaligenes,Rhodopseudomonas palustris, Spingomonas sp.A1, Sphingomonas paucimobilisSYK-6, Sphingomonas japonicum, Sphingomonas alaskenesis, Sphingomonaswittichii, Streptomyces viridosporus, Delftia acidivorans, andRhodococcus equi. Both bio-informatic and experimental data from theliterature reveal the presence of extensive metabolic activity towardsaromatic compounds in these strains, making them relevant species forthe discovery of enzymes that hydrolyze lignin-derived oligomers, andfor biotransformations of lignin core structures. Without intending tobe bound by any theory or mechanism of action, these species exhibit,for example, metabolism of aromatic compounds such as benzoate; amino-,fluoro-, and chloro-benzoates; biphenyl; toluene and nitrotoluenes;xylenes; alkylbenzenes; styrene; atrazine; caprolactam; and polycyclicaromatic hydrocarbons.

The microbes can be grown in a fermentor, for example, using methodsknown to one of skill. The enzymes used in the bioprocessing areobtained from the microbes, and they can be intracellular,extracellular, or a combination thereof. As such, the enzymes can berecovered from the host cells using methods known to one of skill in theart that include, for example, filtering or centrifuging, evaporation,and purification. In some embodiments, the method can include breakingopen the host cells using ultrasound or a mechanical device, removedebris and extract the protein, after which the protein can be purifiedusing, for example, electrophoresis. In some embodiments, however, theteachings include the use of a microbe, recombinant or non-recombinant,that has tolerance to lignin-derived compounds. A microbe that istolerant to lignin-derived compounds can be used industrially, forexample, to express any enzyme, recombinant or non-recombinant, having adesired enzyme activity while directly in association with thelignin-derived compounds. Such activities include, for example, betaetherase activity, C-alpha-dehydrogenase activity, glutathione lyaseactivity, or any other enzyme activity that would be useful in thebiotransformation of lignin-derived compounds. The activities can bewild-type or produce through methods known to one of skill, such astransfection or transformation, for example.

Microbial Systems—Azotobacter Strains

The teachings herein are also directed to the discovery and use ofrecombinant Azotobacter strains heterologously expressing novelbeta-etherase enzymes for the hydrolysis of lignin oligomers.

Research directed to the discovery of a suitable microbe has shown thatAzotobacter vinelandii may possess the industrially relevant straincriteria desired for the teachings provided herein. In some embodiments,the criteria includes (i) growth on inexpensive and defined medium, (ii)resistance to inhibitors in hydrolysates of lignocellulose, (iii)tolerance to acidic pH and higher temperatures, (iv) the co-fermentationof pentose and hexose sugars, (v) genetic tractability and availabilityof gene expression tools, (vi) rapid generation times, and (vii)successful growth performance in pilot scale fermentations.Additionally, key physiological traits that contribute to the potentialsuitability of A. vinelandii to the conversion of lignin-streams includean ability to metabolize aromatic compounds and xenobiotics. Moreover,it has been shown to have a tolerance to phenolic compounds inindustrial waste streams. The annotated genome sequence of A.vinelandii, and the availability of genetic tools for its transformationand for the heterologous expression of enzymes, contribute to thepotential of this microbe to function, in it's native form or as atransformant, for example, in a high-yield production of industrialchemicals from lignin streams.

The teachings are also directed to a method of cleaving a beta-arylether bond, the comprising contacting a polypeptide taught herein with alignin-derived compound having (i) a beta-aryl ether bond and (ii) amolecular weight ranging from about 180 Daltons to about 3000 Daltons;wherein, the contacting occurs in a solvent environment in which thelignin-derived compound is soluble. The term “contacting” refers toplacing an agent, such as a compound taught herein, with a targetcompound, and this placing can occur in situ or in vitro, for example.

The teachings are also directed to a method of cleaving a beta-arylether bond, the comprising contacting a polypeptide taught herein with alignin-derived compound having (i) a beta-aryl ether bond and (ii) amolecular weight ranging from about 180 Daltons to about 3000 Daltons;wherein, the contacting occurs in a solvent environment in which thelignin-derived compound is soluble. In some embodiments, thelignin-derived compound has a molecular weight of about 180 Daltons toabout 1000 Daltons. In some embodiments, the solvent environmentcomprises water. And, in some embodiments, the solvent environmentcomprises a polar organic solvent.

The teachings are also directed to a system for bioprocessinglignin-derived compounds, the system comprising a polypeptide taughtherein, a lignin-derived compound having a beta-aryl ether bond and amolecular weight ranging from about 180 Daltons to about 3000 Daltons;and, a solvent in which the lignin-derived compound is soluble; wherein,the system functions to cleave the beta-aryl ether bond by contactingthe polypeptide with the lignin-derived compound in the solvent.

The teachings are also directed to a recombinant polynucleotidecomprising a nucleotide sequence that encodes a polypeptide taughtherein. Likewise, the teachings are also directed to a vector or plasmidcomprising the polynucleotide, as well as a host cell transformed by thevector or plasmid to express the polypeptide.

The teachings are also directed to a method of cleaving a beta-arylether bond, the method comprising (i) culturing a host cell taughtherein under conditions suitable to produce a polypeptide taught herein;(ii) recovering the polypeptide from the host cell culture; and, (iii)contacting the polypeptide of claim 1 with a lignin-derived compoundhaving a beta-aryl ether bond and a molecular weight ranging from about180 Daltons to about 3000 Daltons; wherein, the contacting occurs in asolvent environment in which the lignin-derived compound is soluble.

In some embodiments, the host cell can be E. Coli or an Azotobacterstrain, such as Azotobacter vinelandii. And, in some embodiments, thelignin-derived compound can have a molecular weight of about 180 Daltonsto about 1000 Daltons.

The teachings are also directed to a system for bioprocessinglignin-derived compounds, the system comprising (i) a transformed hostcell taught herein; (ii) a lignin-derived compound having a beta-arylether bond and a molecular weight ranging from about 180 Daltons toabout 3000 Daltons; and, (iii) a solvent in which the lignin-derivedcompound is soluble; wherein, the system functions to cleave thebeta-aryl ether bond by contacting a polypeptide taught herein with thelignin-derived compound in the solvent.

EXAMPLES

The following examples illustrate, but do not limit, the presentinvention.

Example 1

Microbial growth and metabolism studies on soluble lignin samples areperformed to test the tolerance of microbes on lignin-derived compounds.A set of aromatic and nonaromatic compounds known to inhibit growth ofE. coli and S. cerevisiae strains might be used to characterize thegrowth, tolerance and metabolic capability of Azotobacter vinelandiistrain BAA1303, and A. chroococcum strain 4412 (EB Fred) X-50.Metabolism of various aromatic and nonaromatic compounds by microbialstrains might be determined as a function of cellular respiration by thereduction of soluble tetrazolium salts by actively metabolizing cells.XTT(2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilideinner salt, Sigma) is reduced to a soluble purple formazan compound byrespiring cells. E. coli might be used as the negative control strain inthis study. Strains might be grown in rich medium to saturation, washed,and OD600 nm of the cultures determined. Equal numbers of bacteria willbe inoculated into wells of the 48-well growth asing concentrations ofaromatic and non-aromatic compounds in the range of 0-500 mM, will beadded to the wells to a final volume of 0.8 ml. Following incubation for24-48 hours with shaking at 25-37° C., the cultures will be tested forgrowth upon exposure to the test compounds using the XTT assay kit(Sigma). Culture samples will removed from the 48 well growth plate, anddiluted appropriately in 96 well assay plates to which the XTT reagentwill be added. Soluble formazan formed will be quantified by absorbanceat 450 nm. Increased absorbance at 450 nm will be indicative of growthor survival, or metabolism of a particular test compound by the strains.Table 3 lists some example compounds that can be used to test thetolerance of microbes on lignin-derived compounds.

TABLE 3 Test Compound 1 Syringic acid 2 Syringaldehyde 3 Gallic acid 4Furfural 5 5-Hydroxymethylfurfural 6 4-hydroxybenzaldehyde 7Hydroxybenzoate 8 Vanillin 9 Vanillic acid 10 Cinnamic acid 11 o-, m-andp-Coumaric acids 12 2-hydroxy-3-methoxybenzaldehyde 132,4,6-trihydroxybenzaldehyde 14 4-hydroxy-2,6-dimethoxybenzaldehyde

The set of lignin compounds to be tested might be expanded to any of theteachings provided herein. And, the microbial growth and metabolismstudies on soluble lignin samples can also be performed actualindustrial samples such as, for example, kraft lignins and biorefinerylignins.

Example 2

This example illustrates how prospective enzymes were identified for usewith the teachings provided herein. Although never successfullyexpressed heterologously as an industrial microbe in a commercial scaleprocess, Sphingomonas paucimobilis has been shown to produce enzymesthat have some activity in cleaving the beta aryl ether bond in lignin.See Masai, E., et al. Accordingly, the enzyme discovery effort startedwith running BLAST searches against the two enzymes identified by Masaias having beta etherase activity, “ligE” and “ligF”. See Id. atAbstract. Table 4 lists genes identified in the BLAST searches forinitial screening.

TABLE 4 Genbank Identity/Similarity Gene Species Activity Accession #(%) 1 ligE Sphingomonas Beta-etherase BAA02032.1 paucimobilis 2 ligE-1Novosphingobium Putative ABD26841.1 (62%) (75%) aromaticivoransBeta-etherase 3 ligF Sphingomonas Beta-etherase BAA02031.1 paucimobilis4 ligF-1 Novosphingobium Putative ABD26530.1 (60%) (77%) aromaticivoransBeta-etherase 5 ligF-2 Novosphingobium Putative ABD27301.1 (47%) (59%)aromaticivorans Beta-etherase 6 ligF-3 Novosphingobium PutativeABD27309.1 (37%) (57%) aromaticivorans Beta-etherase

The nucleotide and amino acid sequences in Table 4 are incorporatedherein by reference in their entirety through the GenBank AccessionNumbers.

Example 3

This example describes a method for preparing recombinant host cells forthe heterologous expression of known and putative beta-etherase encodinggene sequences in Escherichia coli (E. coli). E. coli is used in thisexample as a surrogate enzyme production host organism for the enzymediscovery. The construction of a novel industrial host microbe, A.vinelandii is described below.

The gene sequences with accession numbers in Table 3 were synthesizeddirectly as open reading frames (ORFs) from oligonucleotides by usingstandard PCR-based assembly methods, and using the E. coli codon biaswith 10% threshold. The end sequences contained adaptors (NdeI and XhoI)for restriction digestion and cloning into the E. coli expression vectorpET24b (Novagen). Internal NdeI and XhoI sites were excluded from theORF sequences during design of the oligonucleotides. Assembled geneswere cloned into a cloning vector (pGOV4), transformed into E. coli CH3chemically competent cells, and DNA sequences determined from purifiedplasmid DNA. After sequence verification, restriction digestion was usedto excise each ORF fragment from the cloning vector, and the sequencesub-cloned into pET24b. The entire set of ligE and ligF bearing plasmidswere then transformed into E. coli BL21 (DE3) which served as the hoststrain for beta-etherase expression and biochemical activity testing.

LigE, from Accession No BAA2032.1, is listed herein as SEQ ID NO:1 forthe protein and SEQ ID NO:2 for the gene. An “optimized” nucleic acidsequence was created to facilitate the transformation in E. coli and islisted herein as SEQ ID NO:977.

LigE-1, from Accession No ABD26841.1, is listed herein as SEQ ID NO:101for the protein and SEQ ID NO:102 for the gene. An “optimized” nucleicacid sequence was created to facilitate the transformation in E. coliand is listed herein as SEQ ID NO:978.

LigF, from Accession No BAA2031.1 (P30347.1), is listed herein as SEQ IDNO:513 for the protein and SEQ ID NO:514 for the gene. An “optimized”nucleic acid sequence was created to facilitate the transformation in E.coli and is listed herein as SEQ ID NO:979.

LigF-1, from Accession No ABD26530.1, is listed herein as SEQ ID NO:539for the protein and SEQ ID NO:540 for the gene. An “optimized” nucleicacid sequence was created to facilitate the transformation in E. coliand is listed herein as SEQ ID NO:980.

LigF-2, from Accession No ABD27301.1, is listed herein as SEQ ID NO:541for the protein and SEQ ID NO:542 for the gene. An “optimized” nucleicacid sequence was created to facilitate the transformation in E. coliand is listed herein as SEQ ID NO:981.

LigF-3, from Accession No ABD27309.1, is listed herein as SEQ ID NO:545for the protein and SEQ ID NO:546 for the gene. An “optimized” nucleicacid sequence was created to facilitate the transformation in E. coliand is listed herein as SEQ ID NO:982.

Example 3

This example describes a method for gene expression in E. coli, as wellas beta-etherase biochemical assays. Expression of known and putativebeta-etherase genes was performed using 5 ml cultures of the recombinantE. coli strains described herein in Luria Broth medium by induction ofgene expression using isopropylthiogalactoside (IPTG) to a finalconcentration of 0.1 mM. Following induction, and cell harvest, thecells were disrupted using either sonication or the BPER (Invitrogen)cell lysis system.

Clarified cell extracts were tested in the in vitro biochemical assayfor beta-etherase activity on a fluorescent substrate, a model lignindimer compound α-O-(β-methylumbelliferyl) acetovanillone (MUAV). Invitro reactions were performed in a total volume of 200 ul andcontained: 25 mM TrisHCl pH 7.5; 0.5 mM dithiothreitol; 1 mMglutathione; 0.05 mM or 0.1 mM MUAV; 10 ul of clarified cell extractused to initiate the reactions. Following incubation for 2.5 hours atroom temperature, a 50 ul sample of the reactions was terminated using150 uL of 300 mM glycine/NaOH buffer pH 9. The formation of 4methylumbelliferone (4MU) upon hydrolysis of the aryl ether bond wasmonitored by the increase in fluorescence at λ_(ex)=360 nm andλ_(em)=450 nm using a Spectramax UV/visible/fluorescentspectrophotometer.

The total protein concentrations of the cell lysates were determinedusing the BCA reagent system for protein quantification (Pierce).

Induction might be also performed using IPTG concentrations in the rangeof 0.01-1 mM. Cell disruption might be also performed using toluenepermeabilization, French pressure techniques, or using multiplefreeze/thaw cycles in conjunction with lysozyme. Assay conditions mightbe varied to include TrisHCl at 10-150 mM concentrations and in the pHrange of 6.5-8.5; 0-2 mM dithiothreitol; 0.05-2 mM glutathione; 0.01-5mM MUAV substrate; 22-42° C. reaction temperatures. The biochemicalassay might be performed as a fixed time point assay with reaction timesranging from 5 minutes-12 hours, or performed continuously withoutquenching with glycine/NaOH buffer to extract enzyme kinetic parameters.

Example 4

This example describes the tested biochemical activities of thenewly-discovered beta-etherase enzymes.

FIG. 4 illustrates unexpected results from biochemical activity assaysfor beta-etherase function for the S. paucimobilis positive controlpolypeptides, and the N. aromaticivorans putative beta-etherasepolypeptide, according to some embodiments. The much elevatedbeta-etherase activity exhibited by the putative ligE1 gene product fromN. aromaticivorans as compared to the S. paucimobilis ligE gene productwas a completely unexpected result of the enzyme discovery program.

In reactions containing 0.1 mM MUAV substrate, E. coli cell extractsexpressing the N. aromaticovorans ligE1 protein yielded a total activityof 529 rfu/ug compared to 7 rfu/ug for the S. paucimobilis ligE protein.The newly discovered beta-etherase from N. aromaticovorans isapproximately 75-fold more efficient than the previously described S.paucimobilis ligE beta-etherase enzyme. The highly efficient novelbeta-etherase is ideally suited to be a biocatalyst for conversion oflignin aryl ethers to monomers in biotechnological processes.

It was also surprising to find that 3 novel N. aromaticivoranspolypeptides having identities to the S. paucimobilis LigF sequenceshowed beta-etherase activity on the MUAV substrate. While all 3putative ligF gene products from N. aromaticivorans exhibitedbeta-etherase activity, the LigF2 polypeptide is approximately 2-foldmore efficient than the S. paucimobilis LigF protein. The N.aromaticovorans LigF2 protein yielded a total activity of 1206 rfu/ugcompared to 558 rfu/ug for the S. paucimobilis LigF protein.

As such, the enzyme discovery program unexpectedly and surprisinglygenerated four (4) novel polypeptides from N. aromaticivorans withbeta-etherase activity. This set of enzymes show great potential for thecatalysis of a complete depolymerization of lignin-derived compounds.The results were unexpected and surprising for at least the followingreasons:

Four (4) novel gene sequences encoding polypeptides with beta-etheraseactivity were discovered from N. aromaticivorans. These sequences haveGenBank Nos. ABD26841.1 (SEQ ID NO:101); ABD26530.1 (SEQ ID NO:539);ABD27301.1 (SEQ ID NO:541); and ABD27309.1 (SEQ ID NO:545).

One of skill will appreciate that the bioinformatic screen that was usedto help identify putative enzymes is not a definitive predictor initself of biochemical activities, particularly in view of (i) havingonly one known active enzyme for LigE in a different species, (ii) oneknown active enzyme for LigF, and (iii) the unexpected extent of suchactivities discovered. The tests for function therefore had to beperformed empirically on the N. aromaticivorans putative beta-etherasegene set.

One of skill will also appreciate that the discovery of beta-etheraseactivities for all 4 N. aromaticivorans polypeptides was a completesurprise given the relatively low levels of identities (37%-62%) thesequences had with respect to the S. paucimobilis LigE and LigFproteins.

One of skill will also appreciate that the discovery of 2 novelbeta-etherases from the N. aromaticivorans with improved activities overthe corresponding LigE and LigF proteins from S. paucimobilis werecompletely unexpected, and this exciting discovery provides a foundationfor further enzyme development for industrial applications.

Example 5

This example describes the extended use of bioinformatics to identify apool of putative enzymes in the discovery program. As noted above, thebioinformatic screen that was used to help identify putative enzymesinitially was not a definitive predictor in itself of biochemicalactivities, particularly in view of (i) having only one known activeenzyme for LigE in a different species, (ii) one known active enzyme forLigF, and (iii) the unexpected extent of such activities discovered.Having the additional known active enzymes provided more informationthat could be used to enhance the effectiveness of the bioinformatics inidentifying the pool of putative enzymes for both LigE-type andLigF-type enzymes.

Sequence to function correlations for the newly discoveredbeta-etherases were analyzed and identified. A bioinformatic survey offunctional domains, essential catalytic residues, and sequencealignments was performed for the N. aromaticivorans LigE and LigFpolypeptides. While not intending to be bound by any theory or mechanismof action, the rationale and key results of the survey include at leastthe following:

Identifying Functional Domains

As shown in FIG. 4, high levels of beta-etherase activities werediscovered for the N. aromaticivorans LigE1 and LigF2 polypeptidesequences compared to the S. paucimobilis LigE and LigF proteins. The N.aromaticivorans LigE1 and LigF2 polypeptide sequences were used as querysequences for the identification of functional domains using theConserved Domain Database (CDD) in GenBank.

The N. aromaticivorans LigE1 polypeptide is annotated as a glutathioneS-transferase (GST)-like protein with similarity to the GST_C family,and the beta-etherase LigE subfamily. The LigE sub-family is composed ofproteins similar to S. paucimobilis beta etherase, LigE, a GST-likeprotein that catalyzes the cleavage of the beta-aryl ether linkagespresent in low-moleculer weight lignins using reduced glutathione (GSH)as the hydrogen donor in the reaction. The GST fold contains anN-terminal thioredoxin-fold domain and a C-terminal alpha helicaldomain, with an active site located in a cleft between the two domains.

Table 5 describes conserved domains and essential amino acid residues inthe N. aromaticivorans LigE1 polypeptide (ABD26841.1), according to someembodiments. The three (3) conserved functional domains annotated in theN. aromaticivorans LigE1 polypeptide are: i) the dimer interface; ii)the N terminal domain; iii) the lignin substrate binding pocket or the Hsite. Amino acid residues defining the functional domains in suchembodiments are residues 98-221 in the N. aromaticivorans LigE1polypeptide.

Table 5 also lists fifteen (15) amino acid residues as conserved andessential for catalytic activity (column 3 of Table 5), according tosome embodiments. These include: K100; A101; N104; P166; W107; Y184;Y187; R188; G191; G192; F195; V111; G112; M115; F116. While notintending to be bound by any theory or mechanism of action, theseresidues appear responsible for the high beta-etherase catalyticactivity discovered for the N. aromaticivorans LigE1 polypeptidecompared to the S. paucimobilis ligE polypeptide.

In such embodiments, the essential amino acid residues of the N.aromaticivorans LigE1 polypeptide might be altered conservatively, andsingly or in combination with similar amino acid residues that wouldretain or improve the catalytic function of the N. aromaticivorans LigE1polypeptide. Examples of such alternate residues that might beincorporated at the essential positions are also shown in column 4 ofTable 5.

TABLE 5 Residues Conserved defining the residues essential domain in forcatalysis in Alternate residues Functional N. aromaticivorans N.aromaticivorans suggested domain LigE1 LigE1 for the essential positionsDimer interface (residues 98-221 of K100; A101; N104; K100->R SEQ ID NO:101) P166 A101->L; I; V; G; S N104->Q; H; S; A N terminal (residues98-221 of K100; W107; Y184; K100->R domain interface SEQ ID NO: 101)Y187; R188; G191; W107->Y; F; A; S F195 Y184->W; F; A; S Y187-> W; F; A;S R188->K G191-> L; I; V; A; S F195->W; Y; A; S Lignin/substrate(residues 98-221 of W107; V111; G112; W107->Y; F; A; S binding pocket orSEQ ID NO: 101) M115; F116; G192; V111-> L; I; G; A; S H site F195G112-> L; I; V; A; S M115->S; A; G G192-> L; I; V; A; S F195-> W; Y; A;S

The N. aromaticivorans LigF2 polypeptide is annotated as a glutathioneS-transferase (GST)-like protein with similarity to the GST_C family,catalyzing the conjugation of glutathione with a wide range ofxenobiotic agents.

Table 6 describes conserved domains and essential amino acid residues inthe N. aromaticivorans LigF2 polypeptide (ABD27301.1), according to someembodiments. The three (3) conserved functional domains annotated forthe N. aromaticivorans LigF2 polypeptide are similar to those describedfor the N. aromaticivorans LigE polypeptide and comprise: i) the dimerinterface; ii) the N terminal domain; iii) the substrate binding pocketor the H site. In such embodiments, amino acid residues defining thefunctional domains are residues 99-230 in the N. aromaticivorans LigF2polypeptide.

Table 6 also lists sixteen (16) amino acid residues as conserved andessential for catalytic activity (column 3 of Table 6) of the N.aromaticivorans LigF2 polypeptide, according to some embodiments. Theseinclude: R100; Y101; K104; K176; D107; L194; 1197; N198; S201; M206;M111; N112; S115; M116; M206; H202. While not intending to be bound byany theory or mechanism of action, these 16 residues appear to beresponsible for the high beta-etherase catalytic activity discovered forthe N. aromaticivorans LigF2 polypeptide compared to the S. paucimobilisLigF polypeptide.

In such embodiments, the essential amino acid residues of the N.aromaticivorans LigF2 polypeptide might be altered conservatively, andsingly or in combination with similar amino acid residues that wouldretain or improve the catalytic function of the N. aromaticivorans LigF2polypeptide. Examples of such alternate residues that might beincorporated at the essential positions are shown in column 4 of Table6.

TABLE 6 Residues Conserved defining the domain residues essential in forcatalysis in Alternate residues Functional N. aromaticivorans N.aromaticivorans suggested domain LigF2 LigF2 for the essential positionsDimer interface (residues 99-230 of R100; Y101; K104; R100->K SEQ ID NO:541) K176 Y101-> W; F; A; S K104->R K176->R N terminal (residues 99-230of R100; D107; L194; R100->K domain SEQ ID NO: 541) I197; N198; S201;D107->E interface M206 L194-> V; I; G; A; S I197-> L; V; G; A; S N198->QS201->A; M; G M206->S; A; G Substrate (residues 99-230 of D107; M111;N112; D107->E binding pocket SEQ ID NO: 541) S115; M116; M206; M111->S;A; G or H site H202 N112->Q S115->A; M; G M116->S; A; G M206->S; A; GH202->N; Q; S; M

Identifying Additional Functional Domains

Bioinformatic methods were used to further understand the proteinstructure that may result in the desired activities. First, the LigE1and LigF2 were analyzed together. Amino acid sequence alignments wereperformed using the N. aromaticivorans ligE1 (ABD26841.1) and ligF2(ABD27301.1) sequences using the BLAST-P program in GenBank, and thePropom and PraLine programs. Full length sequence alignments yieldedhits with relatively low identities, for example, identities of <70%.

Next, regions in LigE1 and LigF2 were analyzed independently in GENBANK.For LigE1, an alignment was performed against the database in GENBANKusing the following query sequence:“tispfvwatkyalkhkgfdldvvpggftgilertgg” (residues 19-54 of SEQ IDNO:101), from N. aromaticivorans ligE1. The BLAST yielded at least 3subject sequences with high identities in the thioredoxin (TRX)-likesuperfamily of proteins containing a TRX fold. Many members contain aclassic TRX domain with a redox active CXXC motif.

Without intending to be bound by any theory or mechanism of action, theyare thought to function as protein disulfide oxidoreductases (PDOs),altering the redox state of target proteins via the reversible oxidationof their active site dithiol. The PDO members of this superfamilyinclude the families of TRX, protein disulfide isomerase (PDI), tlpA,glutaredoxin, NrdH redoxin, and bacterial Dsb proteins (DsbA, DsbC,DsbG, DsbE, DsbDgamma). Members of the superfamily that do not functionas PDOs but contain a TRX-fold domain include phosducins,peroxiredoxins, glutathione (GSH) peroxidases, SCO proteins, GSHtransferases (GST, N-terminal domain), arsenic reductases, TRX-likeferredoxins and calsequestrin, among others.

Table 7 lists 3 subject sequences having high identities (>80%) toresidues 19-54 of LigE-1 (SEQ ID NO:101). In some embodiments, thesesequences are likely to be essential to catalytic functions similar tothose discovered for the N. aromaticivorans ligE1 polypeptide.

TABLE 7 Identity/Similarity to N. aromaticovorans LigE1 query GenBanksequence residues Subject sequence Species; Gene accession # 19-54 (%)(residues 19-54 of Sphingomonas BAA02032.1 89/97 SEQ ID NO: 1)paucimobilis; beta TISPYVWRTKYALKHKGFDI etherase DIVPGGFTGILERTGG(residues 19-54 of Novosphingobium sp. YP004533906.1 86/92SEQ ID NO: 89) PP1Y; glutathione S TISPFVWRTKYALAHKGFDtransferase like protein VDIVPGGFTGIAERTGG (residues 19-54 ofSphingobium sp. SYK-6; BAJ11989.1 83/94 SEQ ID NO: 3) beta-etheraseTISPFVWATKYAIAHKGFEL DIVPGGFSGIPERTGG

The nucleotide and amino acid sequences in Table 7 are incorporatedherein by reference in their entirety through the GenBank AccessionNumbers.

Likewise, for LigF2, separate alignments were performed against thedatabase in GENBANK using the following 2 query sequences: “ainpegqvpvl”(residues 47-57 of SEQ ID NO:541); and “iithttvineyled” (residues 63-76of SEQ ID NO:541), from N. aromaticivorans ligF2 (ABD27301.1) yieldedmultiple subject sequences with high identities in the GST-N superfamilyof proteins. Without intending to be bound by any theory or mechanism ofaction, the N terminal region (residues 43-75 of SEQ ID NO:541) of theN. aromaticivorans ligF2 polypeptide is annotated in the CDD toencompass:

i. N terminal residues thought to make contact with the C terminalinterface in forming the tertiary protein structure for the GST-N familyof proteins;

ii. N terminal residues thought to be involved in dimerization of thepolypeptides; and,

iii. Residues thought to be involved in the binding of glutathionesubstrate.

Table 8 provides the percent identities and similarities to N.aromaticovorans LigF2 query sequence residues 47-57.

TABLE 8 Identity/Similarity to N. aromaticovorans LigF2 GenBankquery sequence Subject sequence Species; Gene accession #residues 47-57 (%) (residues 45-55 of Proteus mirabilis ATCCZP_03840063.1 91/91 SEQ ID NO: 983) 29906; glutathione S- AINPKGQVPVLtransferase (residues 60-70 of Neisseria macacae ATCC ZP_08683997.182/91 SEQ ID NO: 985) 33926; glutathione S- AINPQGQVPAL transferase(residues 43-53 of Rhodospirillum rubrum; YP_425114.1 82/91SEQ ID NO: 987) glutathione S-transferase- AMNPEGEVPVL like protein(residues 46-56 of Neisseria sicca ATCC ZP_05317369.1 82/91SEQ ID NO: 989) 29256; glutathione S- AINPQGQVPAL transferase(residues 46-56 of Neisseria mucosa ATCC ZP_05978410.1 82/91SEQ ID NO: 991) 25996; glutathione S- AINPQGQVPAL transferase(residues 19-29 of alpha proteobacterium ZP_02189431.1 82/91SEQ ID NO: 993) BAL199; Glutathione S- AINPAGEVPVLtransferase-like protein (residues 31-41 of Marinomonas sp. MED121;ZP_01077889.1 91/91 SEQ ID NO: 995) glutathione S-transferaseAINPLGQVPVL (residues 46-55 of Proteus penneri ATCC ZP_03805830.1 90/90SEQ ID NO:997) 35198; hypothetical protein INPKGQVPVL PROPEN_04226(residues 45-55 of AURANDRAFT_7474 EGB13094.1 82/91 SEQ ID NO: 999)Aureococcus AINPQGKVPVL anophagefferens; hypothetical protein

The nucleotide and amino acid sequences in Table 8 are incorporatedherein by reference in their entirety through the GenBank AccessionNumbers.

Table 9 provides the percent identities and similarities to N.aromaticovorans LigF2 query sequence residues 63-76.

TABLE 9 Identity/Similarity to N. aromaticovorans GenBankLigF2 query sequence Subject sequence Species; Gene accession #residues 63-76 (%) (residues 107-115 of Trichophyton verrucosumXP_003019921.1 100/100 SEQ ID NO: 1001) HKI 0517; conserved TVINEYLEDhypothetical protein (residues 103-111 of Arthroderma benhamiaeXP_003017304.1 100/100 SEQ ID NO: 1003) CBS 112371; conserved TVINEYLEDhypothetical protein (residues 72-80 of Trichophyton rubrum CBSXP_003232549.1 100/100 SEQ ID NO: 1005) 118892; glutathione TVINEYLEDtransferase (residues 62-75 of Novosphingobium sp. PP1Y; YP_004533905.179/79 SEQ ID NO: 1007) glutathione S-transferase- IITESTVICEYLEDlike protein (residues 84-92 of Arthroderma gypseum CBS XP_003171868.1 89/100 SEQ ID NO: 1009) 118893; hypothetical protein TVINEFLEDMGYG_06412 (residues 61-69 of Trichophyton equinum CBS EGE04518.1 89/100 SEQ ID NO: 1011) 127.97; hypothetical protein TVINEFLEDTEQG_03389

The nucleotide and amino acid sequences in Table 9 are incorporatedherein by reference in their entirety through the GenBank AccessionNumbers.

The bioinformatics provides valuable information about protein structurethat can assist in identifying test candidates. For example, the LigE1has the 98-221 region, which is annotated in the databases aspotentially responsible as component of binding and activity,dimerization, and for binding and catalysis in general. While notintending to be bound by any theory or mechanism of action, thevariability in active site structures is reflected by the variability insubstrate structures. Likewise, upon further research usingbioinformatics, it was further discovered that the 19-54 region, whichis annotated in the databases as a second region that is potentiallyresponsible as component of the reductase function, and thus potentiallyresponsible for catalysis in addition to the 98-221 region, while havingmore conservation between members.

Obtaining additional structural information that will assist in findinghigh performing proteins within each family of strains is within thescope of the teachings to the extent that the methodology is known toone of skill. A variety of research techniques are known to one ofskill. Bioinformatic methods, such as motif finding, are an example ofone way to obtain the additional structural information. Motif finding,also known as profile analysis, constructs global multiple sequencealignments that attempt to align short conserved sequence motifs amongthe sequences in the query set. This can be done, for example, by firstconstructing a general global multiple sequence alignment, after whichhighly conserved regions are isolated, in a manner similar to what istaught herein, and used to construct a set of profile matrices. Theprofile matrix for each conserved region is arranged like a scoringmatrix but its frequency counts for each amino acid or nucleotide ateach position are derived from the conserved region's characterdistribution rather than from a more general empirical distribution. Theprofile matrices are then used to search other sequences for occurrencesof the motif they characterize.

LigE-1 and LigF-2 were further examined by comparing their structures toother polypeptides of the LigE-type and LigF-type, respectively. Table10A shows conserved residues between the polypeptide sequences of LigEand LigE-1, and Table 10B shows shows conserved residues between thepolypeptide sequences of LigF and LigF-2.

TABLE 10A Res Pos M 1 A 2 N 4 N 5 T 6 I 7 T 8 Y 10 D 11 L 12 L 14 G 17 T19 I 20 S 21 P 22 V 24 W 25 T 27 K 28 Y 29 A 30 L 31 K 32 H 33 K 34 G 35F 36 D 37 D 39 V 41 P 42 G 43 G 44 F 45 T 46 G 47 I 48 L 49 E 50 R 51 T52 G 53 G 54 E 57 R 58 P 60 I 62 V 63 D 64 D 65 G 66 E 67 V 69 L 70 D 71S 72 W 73 I 75 E 77 Y 78 L 79 D 80 K 82 Y 83 P 84 D 85 R 86 P 87 L 89 K100 L 102 D 103 N 104 W 105 W 107 A 110 V 111 G 112 P 113 W 114 C 117 D121 Y 122 D 124 L 125 S 126 L 127 P 128 Q 129 D 130 Y 133 V 134 S 137 R138 E 139 L 148 E 149 V 151 Q 152 A 153 G 154 R 155 E 156 R 158 L 159 P160 L 166 E 167 P 168 R 170 L 173 A 174 W 178 L 179 G 180 G 181 P 184 N185 A 187 D 188 Y 189 T 198 A 199 S 200 V 201 T 204 P 205 L 207 D 210 D211 P 212 L 213 R 214 D 215 W 216 R 219 D 222 L 223 G 226 L 227 G 228 R229 H 230 P 231 G 232 P 235 L 236 F 237 G 238 L 239 R 242 E 243 G 244 D245 P 246 F 249 R 251 G 254 G 257 N 264 G 266 P 267 T 270 R 275 E 278

As can be seen, there is a high degree of between-species similaritybetween LigE and LigE-1 in the LigE-type family. The LigE residues arefrom S. paucimobilis (BAA02032.1) and the LigE-1 residues are from N.aromaticivorans LigE1 (ABD26841.1). The numbering is done according tothe S. paucimobilis sequence (BAA02032.1) in the PRALINE alignment file(gaps not included).

TABLE 10B Res Pos M 1 Y 6 P 10 A 12 N 13 S 14 K 16 L 21 E 23 K 24 G 25 L26 E 29 D 34 F 38 E 39 H 41 F 45 I 48 N 49 P 50 G 52 V 54 P 55 T 65 T 68I 70 E 72 Y 73 L 74 E 75 D 76 L 85 P 87 D 89 R 97 W 99 K 101 L 161 K 167E 176 L 179 L 185 Y 190 L 192 A 193 D 194 I 195 P 221 L 223 W 226 R 229R 233 P 234 A 235

As can be seen, there is less between-species similarity between LigFand LigF-2 in the LigF-type family. The LigF residues are from S.paucimobilis (BAA02031.1) and the LigF-2 residues are from N.aromaticivorans (ABD27301.1). Numbering is according to the S.paucimobilis sequence (BAA02031.1) in the PRALINE alignment file (gapsnot included.

Example 6

This example provides additional sequences for a second round of assays,the sequences containing the 3 conserved functional domains describedherein for the GST_C family of proteins, and belong to the beta-etheraseLigE subfamily. Table 11 lists nine (9) additional sequences havingidentities of 51%-73% at the amino acid level that were identified inthe SwissProt database using the S. paucimobilis LigE sequence(P27457.3) as the query. The bioinformatics information suggests thatthese 9 sequences are excellent candidates for the next round ofsynthesis, cloning, expression and testing for the desired biochemicalfunctions using the methods described herein.

TABLE 11 Identity to S. paucimobilis Accession # LigE AnnotationSwissProt/GenBank polypeptide (%) 7 Dianthus caryophyllus; Glutathione SP28342.1/121736 59 transferase 8 Euforbua esula; Glutathione SP57108.1/11132235 51 transferase 9 Zea mays; Glutathione S transferaseP04907.4/1170090 70 10 Pseudomonas aeruginosa; P57109.1/11133449 58Maleylacetoacetate isomerase 11 Zea mays; Glutathione S transferaseP46420.2/1170092 63 12 Arabidopsis thaliana; Glutathione SQ8L7C9.1/75329755 61 transferase 13 Arabidopsis thaliana; Glutathione SP42769.1/1170093 73 transferase 14 Oryza sativa Japonica Group;O65857.2/57012737 59 Probable Glutathione S transferase 15 Oryza sativaJaponica Group; O82451.3/57012739 62 Probable Glutathione S transferase

The nucleotide and amino acid sequences in Table 11 are incorporatedherein by reference in their entirety through the GenBank AccessionNumbers.

Example 7

This example describes how native lignin core structures can behydrolyzed by the action of C alpha-dehydrogenases, beta-etherases, andglutathione-eliminating enzymes.

FIG. 5 illustrates beta-aryl-ether compounds to be tested as substratesrepresenting native lignin structures, according to some embodiments.While MUAV was used as a model substrate in the identification of novelbeta-etherase enzymes, additional aryl-ether compounds such as thoseshown in FIG. 5 might be used to assess substrate specificities of thebeta-etherases towards dimers and trimers of aromatic compoundscontaining the beta-aryl ether linkage and representative of nativelignin structures. Higher order oligomers of molecular weights <2000might be synthesized and tested as well. The compounds might be obtainedby custom organic synthesis, as for the fluorescent substrate MUAV.

FIG. 6 illustrates pathways of guaiacylglycerol-β-guaiacyl ether (GGE)metabolism by S. paucimobilis, according to some embodiments. Enzymes inaddition to LigE/F-like beta etherases might be required to hydrolyzenative lignin core structures. The model β-aryl ether compoundguaiacylglycerol-β-guaiacyl ether (GGE) is believed to contain the mainchemical linkages present in native lignin, including the hydroxyl,aryl-ether and methoxy functionalities. The biotransformation of GGE tothe lignin monomer beta-hydroxypropiovanillone (beta-HPV) is partiallyunderstood for S. paucimobilis, and proposed to occur via the action of3 separate enzymes in a step-wise manner. The ligD gene product encodesa C alpha-dehydrogenase which oxidizes GGE toα-(2-methoxyphenoxy)-β-hydroxypropiovanillone (MPHPV); the ether bond ofMPHPV is cleaved by the beta-etherase activities of the ligE and ligFgene products to yield the lignin monomer guaiacol, andα-glutathionylhydroxypropiovanillone (GS-HPV), respectively. The ligGgene product encodes a glutathione (GSH)-eliminating glutathione Stransferase (GST) which catalyzes the elimination of glutathione (GSH)from GS-HPV to yield the lignin hydroxypropiovanillone (HPV).

While the LigE and LigF polypeptides, or similar ones described herein,might be sufficient to hydrolyze native lignin structures, it would beuseful to discover novel C alpha dehydrogenases (S. paucimobilis LigDhomologs) and glutathione (GSH)-eliminating glutathione S transferases(S. paucimobilis LigG homologs) for industrial applications. The enzymediscovery programs might be conducted by methods similar to thosedescribed herein. The detection of lignin substrates, intermediates, andproducts of biochemical reactions might be measured followingfiltration, and the extraction of substrates and products into ethylacetate. Substrates and products might be separated using reverse phaseHPLC conditions with a C18 column developed with a gradient solventsystem of methanol and water, and detected at 230 nm or 254 nm.

Table 12 lists potential C alpha-dehydrogenase polypeptide sequences,the LigD-type, for use in conjunction with beta etherases including, butnot limited to, LigE/F. The sequences were identified usingbioinformatic methods, such as those taught herein. These Calpha-dehydrogenases are classified in the CDD as short-chaindehydrogenase/reductases (SDRs) and are a functionally diverse family ofoxidoreductases that have a single domain with a structurally conservedRossmann fold (alpha/beta folding pattern with a central beta-sheet), anNAD(P)(H)-binding region, and a structurally diverse C-terminal region.Classical SDRs are typically about 250 residues long, while extendedSDRs are approximately 350 residues. Sequence identity between differentSDR enzymes are typically in the 15-30% range, but the enzymes share theRossmann fold NAD-binding motif and characteristic NAD-binding andcatalytic sequence patterns.

Without intending to be bound by any theory or mechanism of action,these enzymes are thought to catalyze a wide range of activitiesincluding the metabolism of steroids, cofactors, carbohydrates, lipids,aromatic compounds, and amino acids, and act in redox sensing. ClassicalSDRs have an TGXXX[AG]XG cofactor binding motif and a YXXXK active sitemotif, with the Tyr residue of the active site motif serving as acritical catalytic residue (Tyr-151, human prostaglandin dehydrogenase(PGDH) numbering). In addition to the Tyr and Lys, there is often anupstream Ser (Ser-138, PGDH numbering) and/or an Asn (Asn-107, PGDHnumbering) contributing to the active site; while substrate binding isin the C-terminal region, which determines specificity.

Without intending to be bound by any theory or mechanism of action, thestandard reaction mechanism is thought to be a 4-pro-S hydride transferand proton relay involving the conserved Tyr and Lys, a water moleculestabilized by Asn, and nicotinamide. Extended SDRs have additionalelements in the C-terminal region, and typically have a TGXXGXXGcofactor binding motif. Complex (multidomain) SDRs such as ketoreductasedomains of fatty acid synthase can have a GGXGXXG NAD(P)-binding motifand an altered active site motif (YXXXN). Fungal type ketoacylreductases can have a TGXXXGX(1-2)G NAD(P)-binding motif. Some atypicalSDRs are thought to have lost catalytic activity and/or have an unusualNAD(P)-binding motif and missing or unusual active site residues.Reactions catalyzed within the SDR family can include isomerization,decarboxylation, epimerization, C═N bond reduction, dehydrataseactivity, dehalogenation, Enoyl-CoA reduction, and carbonyl-alcoholoxidoreduction.

TABLE 12 Identity/Similarity to GenBank Accession S. paucimobilis LigDSpecies Numbers polypeptide (%) 1 N. aromaticivorans YP495487.1 78/88 2N. aromaticivorans YP496072.1 39/58 3 N. aromaticivorans YP496073.139/59 4 N. aromaticivorans YP495984.1 35/56 5 N. aromaticivoransYP497149.1 38/58

The nucleotide and amino acid sequences in Table 12 are incorporatedherein by reference in their entirety through the GenBank AccessionNumbers.

Table 13 lists potential LigG (glutathione-eliminating)-like enzymesequences for use in conjunction with beta etherases including, but notlimited to, LigE/F. The sequences were identified using bioinformaticmethods, such as those taught herein. These might be utilized inconjunction with C-alpha dehydrogenases, and/or with LigE/F-likebeta-etherases. The LigG-like proteins are annotated in the CDD asglutathione S-transferase (GST)-like proteins with similarity to theGST_C family, the GST-N family, and the thioredoxin (TRX)-likesuperfamily of proteins containing a TRX fold.

TABLE 13 Identity/Similarity to GenBank Accession S. paucimobilis LigGSpecies Numbers polypeptide (%) 1 N. aromaticovorans YP_498160.1 23/41 2A. vinelandii DJ YP_002798340 32/50

The nucleotide and amino acid sequences in Table 13 are incorporatedherein by reference in their entirety through the GenBank AccessionNumbers.

Example 8

This example describes the creation of a novel recombinant microbialsystem for the conversion of lignin oligomers to monomers. Azotobactervinelandii strain BAA-1303 DJ, for example, might be transformed withbeta-etherase encoding genes from N. aromaticovorans with the objectiveof creating a lignin phenolics-tolerant A. vinelandii strain capable ofconverting lignin oligomers to monomers at high yields in industrialprocesses. Table 14 lists additional A. vinelandii strains that might beused as host strains for beta-etherase gene expression, for example, bytheir strain designation and American Type Culture Collection (ATCC)number.

TABLE 14 Strain Strain Strain Desig- ATCC Desig- ATCC Desig- ATCC #nation Number # nation Number # nation Number 1 Wiscon- 12518 8 Ad11617962 14 B-6 7489 sin O 2 3a 12837 9 NRS 16 25308 15 B-9 7492 3 AV-313266 10 UWD 478 16 37 9046 4 AV-4 13267 11 113 53800 17 V1 7496 5 AV-513268 12 B-1 7484 18 3 9047 6 OP 13705 13 B-4 7487 7 135 53799 — — — — —— [VKMB- 547]

The heterologous production of beta etherases, Cα dehydrogenases, andother enzymes for the production of lignin monomers and aromaticproducts in A. vinelandii might be achieved using the expression plasmidsystem described herein. The broad host range multicopy plasmid pKT230(ATCC) encoding streptomycin resistance might be used for gene cloning.Genes can be synthesized by methods describe above, and cloned into theSmaI site of pKT230. The nifH promoter from A. vinelandii strain BAA1303 DJ can be used to control gene expression.

A. vinelandii strain BAA 1303 DJ might be transformed with pKT230derivatives using electroporation of electrocompetent cell (Eppendorfmethod), or by incubation of plasmid DNA with chemically competent cellsprepared in TF medium (1.9718 g of MgSO4, 0.0136 g of CaSO4, 1.1 g ofCH3COONH4, 10 g of glucose, 0.25 g of KH2PO4, and 0.55 g of K2HPO4 perliter). Transformants might be selected by screening for resistance tostreptomycin. Gene expression might be induced by cell growth undernitrogen-free Burk's medium (0.2 g of MgSO4, 0.1 g of CaSO4, 0.5 g ofyeast extract, 20 g of sucrose, 0.8 g of K2HPO4, and 0.2 g of KH2PO4,with trace amounts of FeCl3 and Na2MoO4, per liter).

The biochemical activity of a newly-discovered beta-etherase enzymefunctionally expressed in A. vinelandii strain BAA 1303 DJ can be testedusing methods known to one of skill, such as the methods providedherein. Biochemical activity assays for beta-etherase function, and fortotal protein might be performed as described herein.

Example 9

This example describes the design and use of recombinant Azotobacterstrains heterologously expressing enzymes for the production of highvalue aromatic compounds from lignin core structures. Table 15 lists afew examples of aromatic compounds that might be produced by themicrobial platforms described herein.

TABLE 15 Market Volume Market Chemical (metric Value Product ton/year)($/lb) Uses

 30 × 10³  2.34 Antioxidant: 4-tert- butylcatechol. Flavors: piperonal;veratrol. Insecticides: carbofuran; propoxur.

 20 × 10³  6.12 Flavor agent. Precursor for pharmaceutical methyldopa.

  3 × 10⁶  1.65 Precursor to toluene diisocyanates for urethanepolymers.

1.6 × 10³ (US)  3.92 Precursor to analgesic drug acetylsalicylic acid.Precursor to fragrances: amyl and methyl esters of salicylic acid.

57.38 Tuberculosis drug.

 38 × 10³ 0.8 Precursors to herbicides: 4-chloro-2- methylphenoxyaceticacid; 2-(4-chloro-2- methylphenoxy)- propionic acid.

One example of a microbial process to a commercial aromatic compoundmight be the production of catechol from lignin-derived phenoliccompounds. Catechol might be produced from guaiacol using an A.vinelandii or A. chroococcum strain engineered with enzymes includingbeta-etherases and demethylases, or demethylase enzymes alone.Azotobacter strains might be engineered to express the heterologousenzymes by the methods described herein.

FIG. 7 illustrates an example of a biochemical process for theproduction of catechol from lignin oligomers, according to someembodiments. The biochemical processes leading to aromatic products suchas catechol might be designed as 3 unit operations described below:

i) Fractionation of soluble lignin—Concentration or partial purificationof soluble biorefinery lignin fractions or phenolic streams usingmethods known to one of skill.

ii) Biotransformation—The biotransformation of the phenolic substratestream might be carried out in a fed-batch bioprocess using Azotobacterstrains engineered to specifically and optimally convert specificlignin-derived phenolic substrates to the final product, such ascatechol. Corn steep liquor might be used the base medium used in thebiotransformations. The phenolic stream might be introduced in fed-batchmode, at concentrations that will be tolerated by the strains.

iii) Product separation—The product, such as catechol, might be purifiedfrom the aqueous culture broths using standard chemical separationmethods such as liquid-liquid extractions (LLE) with solvents of varyingpolarities applied in a sequential manner.

Additional examples of designed biochemical routes to aromatic productsare described below:

i) lignin-derived syringic acid might be converted to gallic acid via a2-step biochemical conversion using aryl aldehyde oxidases anddemethylases.

ii) Lignin-derived vanillin might be converted to protocatechuic acidvia a 2-step biochemical conversion using aryl aldehyde oxidases anddemethylases.

iii) Lignin-derived vanillin might be converted to catechol via a 3-stepbiochemical conversion using aryl aldehyde oxidases, aromaticdecarboxylases, and demethylases.

iv) Lignin-derived 2-methoxytoluene might be converted to the urethaneprecursor 2,4-diaminotoluene via a 4-step biochemical conversion usingdemethylases, ferulate-5-hydroxylases, 2,4-nitrophenol oxidoreductases,and 2,4-nitrobenzene reductases.

In each case, the specific enzymes might be engineered into A.vinelandii or A. chroococcum strains, for example, and the process mightbe performed using unit operations similar to those described herein forthe biochemical production of catechol.

FIG. 8 illustrates an example of a biochemical process for theproduction of vanillin from lignin oligomers, according to someembodiments. Vanillin can be used as a flavoring agent, and as aprecursor for pharmaceuticals such as methyldopa. Synthetic vanillin,for example, can be produced from petroleum-derived guaiacol by reactionwith glyoxylic acid. Vanillin, however, can also be produced fromlignin-derived β-hydroxypropiovanillone (β-HPV) according to the processscheme indicated in FIG. 8. A 2-step biochemical route to vanillin fromβ-HPV can be achieved using the enzymes 2,4-dihydroxyacetophenoneoxidoreductase, and vanillin dehydrogenase or carboxylic acidreductases, engineered into A. vinelandii.

FIG. 9 illustrates an example of a biochemical process for theproduction of 2,4-diaminotoluene from lignin oligomers, according tosome embodiments. Toluene diisocyanate (TDI) can be used in themanufacture of polyurethanes. For example, 2,4-diaminotoluene (2,4-DAT)is the key precursor to TDI. Diaminotoluenes can be producedindustrially by the sequential nitration of toluene with nitric acid,followed by the reduction of the dinitrotoluenes to the correspondingdiaminotoluenes. Both nitration and reduction reactions yield mixturesof toluene isomers from which the 2,4-DAT isomer is purified bydistillation. The conversion of lignin-derived 2-methoxytoluene to2,4-DAT can be achieved according to the process scheme outlined in FIG.9. 2-methoxytoluene can be converted to 2,4-DAT by A. vinelandiiengineered with 4 enzymes to specifically demethylate, hydroxylate,nitrate and aminate methoxytoluene.

FIG. 10 illustrates process schemes for additional product targets thatinclude ortho-cresol, salicylic acid, and aminosalicylic acid, for theproduction of valuable chemicals from lignin oligomers, according tosome embodiments. These chemicals, as with the others, havetraditionally been obtained from the problematic petrochemicalprocesses. A few of the process schemes for producing these chemicalsusing the teachings herein, based on guaiacol or 2-methoxytoluene, areshown schematically in FIG. 10. Designed biochemical routes, combinedwith the remarkable phenolics-tolerance traits of Azotobacter strainsare proposed for conversions of lignin structures to industrial and finechemicals.

Example 10

This example describes potential LigE-, LigF-, LigG-, and LigD-typepolypeptides, and the genes encoding them. The potential polypeptideswere identified using bioinformatic methods, such as those taughtherein.

As described above, the query sequences in the initial pass for theLigE-type and LigF-type were Sphingomonas paucimobilis sequences, suchas those discussed in Masai, E., et al. Likewise, the query sequencesfor the LigG-type and LigD-type were also Sphingomonas paucimobilissequences, such as those discussed in Masai. The following sequenceswere used in the initial pass for all queries:

LigE, from Accession No BAA2032.1, is listed herein as SEQ ID NO:1 forthe protein and SEQ ID NO:2 for the gene.

LigF, from Accession No BAA2031.1 (P30347.1), is listed herein as SEQ IDNO:513 for the protein and SEQ ID NO:514 for the gene.

LigG, from Accession No Q9Z339.2, is listed herein as SEQ ID NO:733 forthe protein and SEQ ID NO:734 for the gene.

LigD, from Accession No Q01198.1, is listed herein as SEQ ID NO:777 forthe protein and SEQ ID NO:778 for the gene.

The following sequences were used in a modified query to further refinethe LigE-type and LigF-type, and the query sequences were the LigE-1 andLigF-2 that showed the surprising and unexpected results shown in FIG.4:

LigE-1, from Accession No ABD26841.1, is listed herein as SEQ ID NO:101for the protein and SEQ ID NO:102 for the gene.

LigF-2, from Accession No ABD27301.1, is listed herein as SEQ ID NO:541for the protein and SEQ ID NO:542 for the gene.

Table 16 lists SEQ ID NOs:1-246, which are potential protein sequencesof the LigE-type, as well as a respective gene sequence encoding theprotein. Table 17 lists SEQ ID NOs:247-576, which are potential proteinsequences of the LigF-type, as well as a respective gene sequenceencoding the protein. Table 18 lists SEQ ID NOs:577-776, which arepotential protein sequences of the LigG-type, as well as a respectivegene sequence encoding the protein. Table 19 lists SEQ ID NOs: 777-976,which are potential protein sequences of the LigD-type, as well as arespective gene sequence encoding the protein.

Bioinformatic methods, such as those described herein, can be used tosuggest an efficient order of experimentation to identify additionalpotential enzymes for use with the teachings provided herein. Moreover,mutations and amino acid substitutions can be used to test affects onenzyme activity to further understand the structure of the most activeproteins with respect to the enzyme functions sought by teachingsprovided herein.

TABLE 16 PROTEIN GENE GENBANK SEQ ID SEQ ID ACCESSION NO: NO: NO:DESCRIPTION: TYPE 1 2 BAA02032.1 Sphingomonas paucimobilis LIGE 3 4BAJ11989.1 beta-etherase [Sphingobium sp. SYK-6] LIGE 5 6 EFV85608.1glutathione S-transferase domain-containing LIGE protein [Achromobacterxylosoxidans C54] 7 8 EFW42705.1 predicted protein [Capsasporaowczarzaki ATCC LIGE 9 10 EGE55257.1 Glutathione S-transferasedomain-containing LIGE protein [Rhizobium etli CNPAF512] 11 12EGP48556.1 glutathione S-transferase domain-containing LIGE protein[Achromobacter xylosoxidans AXX-A] 13 14 EGP57475.1 lignin degradationprotein [Agrobacterium LIGE 15 16 EGU12703.1 Glutathione S-transferase[Rhodotorula glutinis LIGE ATCC 204091] 17 18 EGU56510.1 glutathioneS-transferase domain-containing LIGE protein [Vibrio tubiashii ATCC19109] 19 20 NP_053324.1 hypothetical protein pTi-SAKURA_p086 LIGE[Agrobacterium tumefaciens] >dbj|BAA87709.1| tiorf84 [Agrobacteriumtumefaciens] 21 22 NP_108131.1 lignin beta-ether hydrolase[Mesorhizobium loti LIGE MAFF303099] >dbj|BAB54276.1|lignin beta- etherhydrolase [Mesorhizobium loti 23 24 NP_354140.2 lignin degradationprotein [Agrobacterium LIGE tumefaciens str. C58] >gb|AAK86925.2|lignindegradation protein [Agrobacterium tumefaciens 25 26 NP_385269.1putative BETA-etherase (BETA-aryl ether LIGE cleaving enzyme) protein[Sinorhizobium meliloti 1021] >emb|CAC45742.1|Putative beta- etherase(beta-aryl ether cleaving enzyme) protein [Sinorhizobium meliloti1021] >gb|AEG03720.1|Glutathione S-transferase domain protein[Sinorhizobium meliloti BL225C] >gb|AEH79753.1|putative BETA-etherase

27 28 NP_774067.1 ligninase [Bradyrhizobium japonicum USDA 110]LIGE >dbj|BAC52692.1|ligE [Bradyrhizobium japonicum USDA 110] 29 30NP_949676.1 putative lignin beta-ether hydrolase LIGE [Rhodopseudomonaspalustris CGA009] >emb|CAE29781.1|putative lignin beta-ether 31 32P27457.3 RecName: Full = Beta-etherase; AltName: LIGE Full = Beta-arylether cleaving enzyme >gb|AAA25878.1|beta-etherase [Sphingomonaspaucimobilis] >dbj|BAA02032.1|beta-etherase 33 34

 P_003028922. 

hypothetical protein SCHCODRAFT_85860 LIGE [Schizophyllum communeH4-8] >gb|EFI94019.1|hypothetical protein 35 36

 P_003030384. 

hypothetical protein SCHCODRAFT_57691 LIGE [Schizophyllum communeH4-8] >gb|EFI95481.1|hypothetical protein 37 38

 P_003033715. 

hypothetical protein SCHCODRAFT_81614 LIGE [Schizophyllum communeH4-8] >gb|EFI98812.1|hypothetical protein 39 40

 P_003041213. 

hypothetical protein NECHADRAFT_55532 LIGE [Nectria haematococca mpVI77-13-4] >gb|EEU35500.1|hypothetical protein NECHADRAFT_55532 [Nectriahaematococca 41 42 XP_382462.1 hypothetical protein FG02286.1[Gibberella zeae LIGE 43 44

 P_001207860. 

putative glutathione S-transferase (GST) LIGE [Bradyrhizobium sp.ORS278] >emb|CAL79645.1|putative glutathione S- 45 46

 P_001236206. 

glutathione S-transferase domain-containing LIGE protein [Acidiphiliumcryptum JF-5] >gb|ABQ32287.1|Glutathione S-transferase, N- terminaldomain protein [Acidiphilium cryptum JF 

47 48

 P_001237901. 

putative glutathione S-transferase LIGE [Bradyrhizobium sp.BTAi1] >gb|ABQ33995.1| putative glutathione S-transferase (GST) 49 50

 P_001262153. 

hypothetical protein Swit_1652 [Sphingomonas LIGE wittichiiRW1] >gb|ABQ68015.1|hypothetical protein Swit_1652 [Sphingomonaswittichii RW1] 51 52

 P_001326465. 

glutathione S-transferase domain-containing LIGE protein [Sinorhizobiummedicae WSM419] >gb|ABR59630.1|Glutathione S-transferase domain[Sinorhizobium medicae WSM419] 53 54

 P_001413220. 

glutathione S-transferase domain-containing LIGE protein [Parvibaculumlavamentivorans DS-1] >gb|ABS63563.1|Glutathione S-transferase domain[Parvibaculum lavamentivorans DS-1] 55 56

 P_001526182. 

glutathione S-transferase [Azorhizobium LIGE caulinodans ORS571] >dbj|BAF89264.1| glutathione S-transferase [Azorhizobium 57 58

 P_001616516. 

lignin degradation protein [Sorangium cellulosum LIGE ‘So ce56’] >emb|CAN96036.1|lignin degradation protein [Sorangium cellulosum‘So 59 60

 P_001772944. 

glutathione S-transferase domain-containing LIGE protein[Methylobacterium sp. 4-46] >gb|ACA20510.1|Glutathione S-transferase 6162

 P_001833458. 

glutathione S-transferase domain-containing LIGE protein [Beijerinckiaindica subsp. indica ATCC 9039] >gb|ACB95969.1|Glutathione S-transferase domain [Beijerinckia indica subsp. 63 64

 P_001977695. 

beta-aryl ether cleaving enzyme, lignin LIGE degradation protein[Rhizobium etli CIAT 652] >gb|ACE90517.1|beta-aryl ether cleavingenzyme, lignin degradation protein [Rhizobium 65 66

 P_001993784. 

glutathione S-transferase domain-containing LIGE protein[Rhodopseudomonas palustris TIE-1] >gb|ACF03309.1|GlutathioneS-transferase domain [Rhodopseudomonas palustris TIE-1] 67 68

 P_002280598. 

glutathione S-transferase domain [Rhizobium LIGE leguminosarum bv.trifolii WSM2304] >gb|ACI54372.1|Glutathione S-transferase domain[Rhizobium leguminosarum bv. trifolii 69 70

 P_002290149. 

glutathione S-transferase [Oligotropha LIGE carboxidovoransOM5] >ref|YP_004631892.1| beta etherase [Oligotropha carboxidovoransOM5] >gb|ACI94284.1|glutathione S- transferase [Oligotrophacarboxidovorans OM5] >gb|AEI02075.1|putative beta etherase [Oligotrophacarboxidovorans OM4]

  71 72

 P_002362903. 

glutathione S-transferase domain-containing LIGE protein [Methylocellasilvestris BL2] >gb|ACK51541.1|glutathione S-transferase 73 74

 P_002502105. 

glutathione S-transferase domain-containing LIGE protein[Methylobacterium nodulans ORS 2060] >gb|ACL61802.1|GlutathioneS-transferase domain protein [Methylobacterium nodulans 75 76

 P_002549116. 

lignin degradation protein [Agrobacterium vitis LIGES4] >gb|ACM36110.1|lignin degradation protein [Agrobacterium vitis S4]77 78

 P_002797805. 

glutathione S-transferase-like protein LIGE [Azotobacter vinelandiiDJ] >gb|ACO76830.1| Glutathione S-transferase-like protein 79 80

 P_002825455. 

putative lignin beta-ether hydrolase LIGE [Sinorhizobium frediiNGR234] >gb|ACP24702.1|putative lignin beta-ether 81 82

 P_002975056. 

glutathione S-transferase domain protein LIGE [Rhizobium leguminosarumbv. trifolii WSM1325] >gb|ACS55517.1|Glutathione S-transferase domainprotein [Rhizobium leguminosarum bv. 83 84

 P_004278359. 

lignin degradation protein [Agrobacterium sp. LIGEH13-3] >gb|ADY64039.1|lignin degradation protein [Agrobacterium sp.H13-3] 85 86

 P_004285673. 

putative beta-etherase [Acidiphilium multivorum LIGEAIU301] >dbj|BAJ82791.1|putative beta- etherase [Acidiphilium multivorumAIU301] 87 88

 P_004378290. 

glutathione S-transferase-like protein LIGE [Pseudomonas mendocinaNK-01] >gb|AEB56538.1|glutathione S-transferase-like 89 90

 P_004533906. 

glutathione S-transferase-like protein LIGE [Novosphingobium sp.PP1Y] >emb|CCA92088.1|glutathione S-transferase- 91 92

 P_004548326. 

glutathione S-transferase domain-containing LIGE protein [Sinorhizobiummeliloti AK83] >gb|AEG52712.1|Glutathione S-transferase 93 94

 P_004613710. 

glutathione S-transferase domain-containing LIGE protein [Mesorhizobiumopportunistum WSM2075] >gb|AEH89616.1|Glutathione S- transferase domainprotein [Mesorhizobium 95 96 YP_269568.1 putative lignin beta-etherase[Colwellia LIGE psychrerythraea 34H] >gb|AAZ24120.1|putative ligninbeta-etherase [Colwellia psychrerythraea 97 98 YP_469001.1 beta-arylether cleaving enzyme, lignin LIGE degradation protein [Rhizobium etliCFN 42] >gb|ABC90274.1|beta-aryl ether cleaving enzyme, lignindegradation protein [Rhizobium 99 100 YP_487746.1 glutathioneS-transferase-like protein LIGE [Rhodopseudomonas palustrisHaA2] >gb|ABD08835.1|Glutathione S-transferase-like 101 102 YP_497675.1glutathione S-transferase-like protein LIGE [Novosphingobiumaromaticivorans DSM 12444] >gb|ABD26841.1|glutathione S-transferase-likeprotein [Novosphingobium aromaticivorans DSM 103 104 YP_533979.1glutathione S-transferase-like protein LIGE [Rhodopseudomonas palustrisBisB18] >gb|ABD89660.1|glutathione S-transferase-like 105 106YP_574731.1 glutathione S-transferase-like protein LIGE[Chromohalobacter salexigens DSM 3043] >gb|ABE60032.1|glutathioneS-transferase-like protein [Chromohalobacter salexigens DSM 107 108YP_723508.1 glutathione S-transferase-like protein LIGE [Trichodesmiumerythraeum IMS101] >gb|ABG53035.1|glutathione S-transferase-like 109 110YP_767183.1 etherase [Rhizobium leguminosarum bv. viciae LIGE3841] >emb|CAK07074.1|putative etherase [Rhizobium leguminosarum bv.viciae 3841] 111 112 YP_783091.1 glutathione S-transferase[Rhodopseudomonas LIGE palustris BisA53] >gb|ABJ08111.1|GlutathioneS-transferase [Rhodopseudomonas palustris 113 114 YP_915395.1glutathione S-transferase domain-containing LIGE protein [Paracoccusdenitrificans PD1222] >gb|ABL69699.1|Glutathione S-transferase, N-terminal domain [Paracoccus denitrificans 115 116 ZP_02146530. 

putative beta-etherase (beta-aryl ether cleaving LIGE enzyme) protein[Phaeobacter gallaeciensis BS107] >gb|EDQ11875.1|putative beta- etherase(beta-aryl ether cleaving enzyme) 117 118 ZP_02149699. 

putative beta-etherase (beta-aryl ether cleaving LIGE enzyme) protein[Phaeobacter gallaeciensis 2.10] >gb|EDQ08644.1|putative beta-etherase(beta-aryl ether cleaving enzyme) protein 119 120 ZP_02166231. 

putative beta-etherase (beta-aryl ether cleaving LIGE enzyme) protein[Hoeflea phototrophica DFL-43] >gb|EDQ33834.1|putative beta-etherase(beta- aryl ether cleaving enzyme) protein [Hoeflea 121 122ZP_02190934. 

glutathione S-transferase-like protein [alpha LIGE proteobacteriumBAL199] >gb|EDP62276.1| glutathione S-transferase-like protein [alpha123 124 ZP_03503368. 

Glutathione S-transferase domain [Rhizobium LIGE 125 126 ZP_03507162. 

Glutathione S-transferase domain [Rhizobium LIGE 127 128 ZP_03513891. 

Glutathione S-transferase domain [Rhizobium LIGE 129 130 ZP_03519388. 

Glutathione S-transferase domain [Rhizobium LIGE 131 132 ZP_03520502. 

putative etherase [Rhizobium etli GR56] LIGE 133 134 ZP_05084767. 

glutathione S-transferase, N-terminal domain LIGE [Pseudovibrio sp.JE062] >gb|EEA94709.1| glutathione S-transferase, N-terminal domain 135136 ZP_06688745. 

lignin degradation protein [Achromobacter LIGE piechaudii ATCC43553] >gb|EFF74366.1|lignin degradation protein [Achromobacterpiechaudii 137 138 ZP_06898146. 

glutathione S-transferase family protein LIGE [Roseomonas cervicalisATCC 49957] >gb|EFH10151.1|glutathione S-transferase family protein[Roseomonas cervicalis ATCC 139 140 ZP_07027473. 

Glutathione S-transferase domain protein [Afipia LIGE sp.1NLS2] >gb|EFI51229.1|Glutathione S- transferase domain protein [Afipiasp. 1NLS2] 141 142 ZP_07373940. 

beta-etherase [Ahrensia sp. R2A130] LIGE >gb|EFL90585.1|beta-etherase[Ahrensia sp. 143 144 ZP_08328512. 

Glutathione S-transferase [gamma LIGE proteobacteriumIMCC1989] >gb|EGG95341.1| Glutathione S-transferase [gamma 145 146ZP_08529965. 

lignin degradation protein [Agrobacterium sp. LIGE ATCC31749] >gb|EGL63395.1|lignin degradation protein [Agrobacterium sp. ATCC147 148 ZP_08627134. 

lignin beta-ether hydrolase [Bradyrhizobiaceae LIGE bacteriumSG-6C] >gb|EGP10168.1|lignin beta- ether hydrolase [Bradyrhizobiaceaebacterium 149 150 ZP_08631370. 

Glutathione S-transferase domain-containing LIGE protein [Acidiphiliumsp. PM] >gb|EGO96849.1| Glutathione S-transferase domain-containing 151152 ZP_08634908. 

Glutathione S-transferase domain-containing LIGE protein [Acidiphiliumsp. PM] >gb|EGO93307.1| Glutathione S-transferase domain-containing 153154 ZP_08635074. 

glutathione S-transferase domain-containing LIGE protein [Halomonas sp.TD01] >gb|EGP21558.1| glutathione S-transferase domain-containing 155156 EGN93792.1 hypothetical protein SERLA73DRAFT_115219 LIGE [Serpulalacrymans var. lacrymans S7.3] >gb|EGO19163.1|hypothetical proteinSERLADRAFT_453680 [Serpula lacrymans var. 157 158 EGN94392.1hypothetical protein SERLA73DRAFT_188253 LIGE [Serpula lacrymans var.lacrymans S7.3] >gb|EGO19875.1|hypothetical protein SERLADRAFT_478300[Serpula lacrymans var. 159 160 EGN96317.1 hypothetical proteinSERLA73DRAFT_186005 LIGE [Serpula lacrymans var. lacrymansS7.3] >gb|EGO21854.1|hypothetical protein SERLADRAFT_474829 [Serpulalacrymans var. 161 162 EGN96924.1 hypothetical proteinSERLA73DRAFT_185168 LIGE [Serpula lacrymans var. lacrymansS7.3] >gb|EGO22516.1|hypothetical protein SERLADRAFT_473468 [Serpulalacrymans var. 163 164 EGO00367.1 hypothetical proteinSERLA73DRAFT_107446 LIGE [Serpula lacrymans var. lacrymansS7.3] >gb|EGO25928.1|hypothetical protein SERLADRAFT_415302 [Serpulalacrymans var. 165 166

 P_001215222. 

conserved hypothetical protein [Aspergillus LIGE terreusNIH2624] >gb|EAU33805.1|conserved hypothetical protein [Aspergillusterreus 167 168

 P_001823934. 

hypothetical protein AOR_1_322094 [Aspergillus LIGE oryzaeRIB40] >dbj|BAE62801.1|unnamed protein product [Aspergillus oryzaeRIB40] 169 170

 P_001839188. 

hypothetical protein CC1G_07903 [Coprinopsis LIGE cinereaokayama7#130] >gb|EAU82621.1| hypothetical protein CC1G_07903[Coprinopsis 171 172

 P_001885678. 

predicted protein [Laccaria bicolor S238N-H82]LIGE >gb|EDR03530.1|predicted protein [Laccaria bicolor S238N-H82] 173174

 P_002152364. 

conserved hypothetical protein [Penicillium LIGE marneffei ATCC18224] >gb|EEA19427.1| conserved hypothetical protein [Penicillium 175176

 P_002380998. 

conserved hypothetical protein [Aspergillus LIGE flavusNRRL3357] >gb|EED49097.1|conserved hypothetical protein [Aspergillusflavus 177 178

 P_002392962. 

hypothetical protein MPER_07394 LIGE [Moniliophthora perniciosaFA553] >gb|EEB93892.1|hypothetical protein 179 180

 P_002468854. 

predicted protein [Postia placenta Mad-698-R]LIGE >gb|EED86077.1|predicted protein [Postia placenta Mad-698-R] 181182

 P_002472522. 

predicted protein [Postia placenta Mad-698-R]LIGE >gb|EED82308.1|predicted protein [Postia placenta Mad-698-R] 183184

 P_002557398. 

Pc12g05530 [Penicillium chrysogenum LIGE Wisconsin54-1255] >emb|CAP80180.1| Pc12g05530 [Penicillium chrysogenum 185 186

 P_003026159. 

hypothetical protein SCHCODRAFT_12387 LIGE [Schizophyllum communeH4-8] >gb|EFI91256.1|hypothetical protein 187 188

 P_003028923. 

hypothetical protein SCHCODRAFT_111982 LIGE [Schizophyllum communeH4-8] >gb|EFI94020.1|hypothetical protein 189 190

 P_003890246. 

Glutathione S-transferase domain-containing LIGE protein [Cyanothece sp.PCC 7822] >gb|ADN16971.1|Glutathione S-transferase 191 192

 P_003896657. 

glutathione S-transferase-like [Halomonas LIGE elongata DSM2581] >emb|CBV41472.1| glutathione S-transferase-like [Halomonas 193 194

 P_003980382. 

glutathione S-transferase [Achromobacter LIGE xylosoxidansA8] >gb|ADP17667.1|glutathione S-transferase, N-terminal domain protein4 195 196

 P_004110838. 

glutathione S-transferase domain-containing LIGE protein[Rhodopseudomonas palustris DX-1] >gb|ADU46105.1|GlutathioneS-transferase domain [Rhodopseudomonas palustris DX-1] 197 198

 P_004143867. 

glutathione S-transferase [Mesorhizobium ciceri LIGE biovar biserrulaeWSM1271] >gb|ADV13817.1| Glutathione S-transferase domain [Mesorhizobiumciceri biovar biserrulae 199 200 ZP_01102591. 

conserved hypothetical protein [Congregibacter LIGE litoralisKT71] >gb|EAQ98305.1|conserved hypothetical protein [Congregibacterlitoralis 201 202 AAA87183.1 auxin-induced protein [Vigna radiata] LIGE203 204 AAG34797.1 glutathione S-transferase GST 7 [Glycine max] LIGE205 206 AAO69664.1 glutathione S-transferase [Phaseolus acutifolius]LIGE 207 208 ACU24385.1 unknown [Glycine max] LIGE 209 210 ADP99065.1glutathione S-transferase [Marinobacter LIGE 211 212 ADY82158.1 putativeglutathione S-transferase [Acinetobacter LIGE calcoaceticus PHEA-2] 213214 BAA77215.1 beta-etherase [Sphingomonas paucimobilis] LIGE 215 216

 P_001839584. 

hypothetical protein CC1G_12612 [Coprinopsis LIGE cinereaokayama7#130] >gb|EAU82225.1| hypothetical protein CC1G_12612[Coprinopsis 217 218

 P_002336443. 

predicted protein [Populus trichocarpa] LIGE >gb|EEE73479.1|predictedprotein [Populus 219 220

 P_003028624. 

hypothetical protein SCHCODRAFT_59314 LIGE [Schizophyllum communeH4-8] >gb|EFI93721.1|hypothetical protein 221 222 XP_456365.1DEHA2A00660p [Debaryomyces hansenii LIGECBS767] >emb|CAG84310.1|DEHA2A00660p [Debaryomyces hansenii] 223 224XP_572781.1 hypothetical protein [Cryptococcus neoformans LIGE var.neoformans JEC21] >ref|XP_773999.1| hypothetical protein CNBH0460[Cryptococcus neoformans var. neoformansB-3501A] >gb|EAL19352.1|hypothetical protein CNBH0460 [Cryptococcusneoformans var. neoformans B-3501A] >gb|AAW45474.1| 225 226

 P_001236206. 

glutathione S-transferase domain-containing LIGE protein [Acidiphiliumcryptum JF-5] >gb|ABQ32287.1|Glutathione S-transferase, N- terminaldomain protein [Acidiphilium cryptum JF 

227 228

 P_001237901. 

putative glutathione S-transferase LIGE [Bradyrhizobium sp.BTAi1] >gb|ABQ33995.1| putative glutathione S-transferase (GST) 229 230

 P_001262153. 

hypothetical protein Swit_1652 [Sphingomonas LIGE wittichiiRW1] >gb|ABQ68015.1|hypothetical protein Swit_1652 [Sphingomonaswittichii RW1] 231 232

 P_001326465. 

glutathione S-transferase domain-containing LIGE protein [Sinorhizobiummedicae WSM419] >gb|ABR59630.1|Glutathione S-transferase domain[Sinorhizobium medicae WSM419] 233 234

 P_001413220. 

glutathione S-transferase domain-containing LIGE protein [Parvibaculumlavamentivorans DS-1] >gb|ABS63563.1|Glutathione S-transferase domain[Parvibaculum lavamentivorans DS-1] 235 236

 P_001526182. 

glutathione S-transferase [Azorhizobium LIGE caulinodans ORS571] >dbj|BAF89264.1| glutathione S-transferase [Azorhizobium 237 238YP_171459.1 glutathione S-transferase [Synechococcus LIGE elongatus PCC6301] >ref|YP_399807.1| glutathione S-transferase [Synechococcuselongatus PCC 7942] >dbj|BAD78939.1| glutathione S-transferase[Synechococcus elongatus PCC 6301] >gb|ABB56820.1| 239 240 YP_322424.1glutathione S-transferase-like protein [Anabaena LIGE variabilis ATCC29413] >gb|ABA21529.1| Glutathione S-transferase-like protein 241 242ZP_01625805. 

glutathione S-transferase, putative [marine LIGE gamma proteobacteriumHTCC2080] >gb|EAW41324.1|glutathione S-transferase, putative [marinegamma proteobacterium 243 244 ZP_01631145. 

Glutathione S-transferase-like protein [Nodularia LIGE spumigenaCCY9414] >gb|EAW44220.1| Glutathione S-transferase-like protein[Nodularia 245 246 ZP_06057261. 

glutathione S-transferase [Acinetobacter LIGE calcoaceticusRUH2202] >gb|EEY78560.1| glutathione S-transferase [Acinetobacter

indicates data missing or illegible when filed

TABLE 17 PROTEIN GENE GENBANK SEQ ID SEQ ID ACCESSION NO: NO: NO:DESCRIPTION: TYPE 247 248 AAB65163.1 glutathione S-transferase,class-phi LigF [Solanum commersonii] 249 250 AAG34850.1 glutathioneS-transferase GST 42 [Zea LigF mays] 251 252 AAK98535.1 putativeglutathione S-transferase LigF OsGSTU7 [Oryza sativa Japonica Group] 253254 AAL61612.1 glutathione S-transferase [Allium cepa] LigF 255 256ABE86679.1 Intracellular chloride channel [Medicago LigF truncatula] 257258 ABE86683.1 Intracellular chloride channel [Medicago LigF truncatula]259 260 ABQ96853.1 glutathione S-transferase [Solanum LigF tuberosum]261 262 ACF15452.1 glutathione-S-transferase LigF [Phanerochaetechrysosporium] 263 264 ACG44597.1 glutathione S-transferase GSTU6 [ZeaLigF mays] 265 266 ACJ86045.1 unknown [Medicago truncatula] LigF 267 268ACO15091.1 Probable maleylacetoacetate isomerase LigF 2 [Caligusclemensi] 269 270 ADB11335.1 phi class glutathione transferase GSTF7LigF [Populus trichocarpa] 271 272 BAB70616.1 glutathione S-transferase[Medicago LigF sativa] 273 274 BAF56180.1 glutathione S-transferase[Allium cepa] LigF 275 276 BAJ90004.1 predicted protein [Hordeum vulgareLigF subsp. vulgare] >dbj|BAJ99460.1| predicted protein [Hordeum vulgaresubsp. vulgare] 277 278 CAI51314.2 glutathione S-transferase GST1 LigF[Capsicum chinense] 279 280 EAY79299.1 hypothetical protein OsI_34425[Oryza LigF sativa Indica Group] 281 282 EAZ16758.1 hypothetical proteinOsJ_32234 [Oryza LigF sativa Japonica Group] 283 284 EEC67342.1hypothetical protein OsI_34397 [Oryza LigF sativa Indica Group] 285 286EFV87279.1 glutathione S-transferase LigF [Achromobacter xylosoxidansC54] 287 288 EGN92742.1 hypothetical protein LigF SERLA73DRAFT_190579[Serpula lacrymans var. lacrymans S7.3] >gb|EGO26403.1|hypotheticalprotein SERLADRAFT_463437 [Serpula lacrymans var. lacrymans S7.9] 289290 EGU75635.1 hypothetical protein FOXB_13869 LigF [Fusarium oxysporumFo5176] 291 292 NP_001065115.1 Os10g0525600 [Oryza sativa Japonica LigFGroup] >gb|AAM12493.1|AC074232_20 putative glutathione S-transferase[Oryza sativa Japonica Group] >dbj|BAF27029.1|Os10g0525600 [Oryza sativaJaponica Group] 293 294 NP_001065118.1 Os10g0527400 [Oryza sativaJaponica LigF Group] >gb|AAM12310.1|AC091680_11 putative glutathioneS-transferase [Oryza sativa Japonica Group] >gb|AAM12478.1|AC074232_5putative glutathione S-transferase [Oryza sativa JaponicaGroup] >gb|AAP54729.1| glutathione S-transferase GSTU6, putative,expressed [Oryza sativa Japonica Group] >dbj|BAF27032.1| Os10g0527400[Oryza sativa Japonica Group] >gb|EEE51298.1|hypothetical proteinOsJ_32225 [Oryza sativa Japonica Group] 295 296 NP_001065126.1Os10g0529300 [Oryza sativa Japonica LigFGroup] >gb|AAK98546.1|AF402805_1 putative glutathione S-transferaseOsGSTU18 [Oryza sativa Japonica Group] >gb|AAM12302.1|AC091680_3putative glutathione S-transferase [Oryza sativa JaponicaGroup] >gb|AAM94529.1|putative glutathione S- transferase [Oryza sativaJaponica Group] >gb|AAP54753.1|glutathione S- transferase GSTU6,putative, expressed [Oryza sativa JaponicaGroup] >dbj|BAF27040.1|Os10g0529300 [Oryza sativa JaponicaGroup] >gb|EAY79288.1|hypothetical protein OsI_34414 [Oryza sativaIndica Group] >dbj|BAG87628.1|unnamed protein product [Oryza sativaJaponica Group] >dbj|BAG97643.1|unnamed protein product [Oryza sativaJaponica Group] >dbj|BAG87189.1|unnamed protein product [Oryza sativaJaponica Group] 297 298 NP_001065132.1 Os10g0529900 [Oryza sativaJaponica LigF Group] >gb|AAM12331.1|AC091680_32 putative glutathioneS-transferase [Oryza sativa Japonica Group] >gb|AAM94517.1|putativeglutathione S- transferase [Oryza sativa JaponicaGroup] >gb|AAP54759.1|glutathione S- transferase GSTU6, putative [Oryzasativa Japonica Group] >dbj|BAF27046.1|Os10g0529900 [Oryza sativaJaponica Group] >gb|EAZ16763.1|hypothetical protein OsJ_32239 [Oryzasativa Japonica Group] 299 300 NP_001105627.1 LOC542632 [Zea mays]LigF >gb|AAG34835.1|AF244692_1 glutathione S-transferase GST 27 [Zeamays] >gb|ACF85142.1|unknown [Zea mays] 301 302 NP_001152229.1glutathione S-transferase GSTU6 [Zea LigFmays] >gb|ACG46501.1|glutathione S- transferase GSTU6 [Zea mays] 303 304NP_384409.1 putative glutathione S-transferase LigF protein[Sinorhizobium meliloti 1021] >ref|YP_004550950.1|glutathione S-transferase domain-containing protein [Sinorhizobium melilotiAK83] >emb|CAC41740.1|Putative glutathione S-transferase [Sinorhizobiummeliloti 1021] >gb|AEG06303.1|Glutathione S- transferase domain protein[Sinorhizobium meliloti BL225C] >gb|AEG55336.1|Glutathione S-transferase domain protein [Sinorhizobium melilotiAK83] >gb|AEH81005.1|putative glutathione S- transferase protein[Sinorhizobium meliloti SM11] 305 306 XP_001555922.1 hypotheticalprotein BC1G_05597 LigF [Botryotinia fuckelianaB05.10] >gb|EDN24875.1|hypothetical protein BC1G_05597 [Botryotiniafuckeliana B05.10] 307 308 XP_001805855.1 hypothetical proteinSNOG_15716 LigF [Phaeosphaeria nodorum SN15] >gb|EAT76811.2|hypotheticalprotein SNOG_15716 [Phaeosphaeria nodorum SN15] 309 310 XP_002321320.1predicted protein [Populus trichocarpa] LigF >gb|EEE99635.1|predictedprotein [Populus trichocarpa] 311 312 XP_002455784.1 hypotheticalprotein LigF SORBIDRAFT_03g025210 [Sorghumbicolor] >gb|EES00904.1|hypothetical protein SORBIDRAFT_03g025210[Sorghum bicolor] 313 314 XP_002467606.1 hypothetical protein LigFSORBIDRAFT_01g030860 [Sorghum bicolor] >gb|EER94604.1|hypotheticalprotein SORBIDRAFT_01g030860 [Sorghum bicolor] 315 316 XP_002734706.1PREDICTED: ganglioside-induced LigF differentiation-associated protein1-like [Saccoglossus kowalevskii] 317 318 XP_002734707.1 PREDICTED:ganglioside-induced LigF differentiation-associated protein 1-like[Saccoglossus kowalevskii] 319 320 XP_002737947.1 PREDICTED: GlutathioneS-Transferase LigF family member (gst-42)-like [Saccoglossuskowalevskii] 321 322 XP_002989538.1 hypothetical protein LigFSELMODRAFT_184606 [Selaginella moellendorffii] >gb|EFJ09414.1|hypothetical protein SELMODRAFT_184606 [Selaginella moellendorffii] 323324 XP_003146962.1 glutathione S-transferase domain- LigF containingprotein [Loa loa] >gb|EFO17107.1|glutathione S- transferasedomain-containing protein [Loa loa] 325 326 YP_001187408.1 glutathioneS-transferase domain- LigF containing protein [Pseudomonas mendocinaymp] >gb|ABP84676.1| Glutathione S-transferase, N-terminal domainprotein [Pseudomonas mendocina ymp] 327 328 YP_001239734.1 glutathioneS-transferase domain- LigF containing protein [Bradyrhizobium sp.BTAi1] >gb|ABQ35828.1|putative glutathione S-transferase enzyme withthioredoxin-like domain [Bradyrhizobium sp. BTAi1] 329 330YP_001261939.1 glutathione S-transferase domain- LigF containing protein[Sphingomonas wittichii RW1] >gb|ABQ67801.1| Glutathione S-transferase,N-terminal domain [Sphingomonas wittichii RW1] 331 332 YP_001263066.1glutathione S-transferase domain- LigF containing protein [Sphingomonaswittichii RW1] >gb|ABQ68928.1| Glutathione S-transferase, N-terminaldomain [Sphingomonas wittichii RW1] 333 334 YP_001414366.1 glutathioneS-transferase domain- LigF containing protein [Parvibaculumlavamentivorans DS-1] >gb|ABS64709.1|Glutathione S- transferase domain[Parvibaculum lavamentivorans DS-1] 335 336 YP_001414838.1maleylacetoacetate isomerase LigF [Parvibaculum lavamentivoransDS-1] >gb|ABS65181.1|maleylacetoacetate isomerase [Parvibaculumlavamentivorans DS-1] 337 338 YP_001684291.1 glutathione S-transferasedomain- LigF containing protein [Caulobacter sp.K31] >gb|ABZ71793.1|Glutathione S- transferase domain [Caulobacter sp.K31] 339 340 YP_001770584.1 glutathione S-transferase domain- LigFcontaining protein [Methylobacterium sp.4-46] >gb|ACA18150.1|Glutathione S- transferase domain [Methylobacteriumsp. 4-46] 341 342 YP_002828116.1 predicted glutathione S-transferaseLigF protein [Sinorhizobium fredii NGR234] >gb|ACP27363.1|predictedglutathione S-transferase protein [Sinorhizobium fredii NGR234] 343 344YP_003593122.1 glutathione S-transferase domain- LigF containing protein[Caulobacter segnis ATCC 21756] >gb|ADG10504.1| GlutathioneS-transferase domain protein [Caulobacter segnis ATCC 21756] 345 346YP_003930867.1 glutathione S-transferase [Pantoea LigF vagansC9-1] >gb|ADO09418.1| Glutathione S-transferase [Pantoea vagans C9-1]347 348 YP_004434596.1 Glutathione S-transferase domain LigF protein[Glaciecola agarilytica 4H-3- 7 + YE-5] >gb|AEE23328.1|Glutathione S-transferase domain protein [Glaciecola sp. 4H-3-7 + YE-5] 349 350YP_004620883.1 glutathione S-transferase [Ramlibacter LigF tataouinensisTTB310] >gb|AEG94864.1|glutathione S- transferase-like protein[Ramlibacter tataouinensis TTB310] 351 352 YP_067874.1 glutathioneS-transferase family protein LigF [Aeromonaspunctata] >emb|CAG15111.1|glutathione S- transferase family protein[Aeromonas caviae] 353 354 YP_168502.1 glutathione S-transferase,putative LigF [Ruegeria pomeroyi DSS-3] >gb|AAV96533.1|glutathione S-transferase, putative [Ruegeria pomeroyi DSS-3] 355 356 YP_339058.1glutathione S-transferase LigF [Pseudoalteromonas haloplanktisTAC125] >emb|CAI85615.1|putative glutathione S-transferase[Pseudoalteromonas haloplanktis TAC125] 357 358 YP_612204.1 glutathioneS-transferase-like [Ruegeria LigF sp. TM1040] >gb|ABF62942.1|glutathione S-transferase-like protein [Ruegeria sp. TM1040] 359 360ZP_00954574.1 glutathione S-transferase family protein LigF[Sulfitobacter sp. EE-36] >ref|ZP_00961889.1|glutathione S- transferasefamily protein [Sulfitobacter sp. NAS-14.1] >gb|EAP81303.1| glutathioneS-transferase family protein [Sulfitobacter sp.NAS-14.1] >gb|EAP85807.1|glutathione S- transferase family protein[Sulfitobacter sp. EE-36] 361 362 ZP_01165363.1 maleylacetoacetateisomerase LigF [Oceanospirillum sp.MED92] >gb|EAR62715.1|maleylacetoacetate isomerase [Oceanospirillum sp.MED92] 363 364 ZP_01881157.1 glutathione S-transferase, putative LigF[Roseovarius sp. TM1035] >gb|EDM30676.1|glutathione S- transferase,putative [Roseovarius sp. TM1035] 365 366 ZP_03523367.1 GlutathioneS-transferase domain LigF [Rhizobium etli GR56] 367 368 ZP_04614975.1Glutathione S-transferase GST-6.0 LigF [Yersinia ruckeri ATCC29473] >gb|EEQ00521.1|Glutathione S- transferase GST-6.0 [Yersiniaruckeri ATCC 29473] 369 370 ZP_05125190.1 glutathione S-transferase,N-terminal LigF domain protein [Rhodobacteraceae bacteriumKLH11] >gb|EEE36118.1| glutathione S-transferase, N-terminal domainprotein [Rhodobacteraceae bacterium KLH11] 371 372 ZP_05786193.1glutathione S-transferase [Silicibacter LigF lacuscaerulensisITI-1157] >gb|EEX09309.1|glutathione S- transferase [Silicibacterlacuscaerulensis ITI-1157] 373 374 ZP_08264339.1 maleylacetoacetateisomerase LigF [Asticcacaulis biprosthecumC19] >gb|EGF90974.1|maleylacetoacetate isomerase [Asticcacaulisbiprosthecum C19] 375 376 ZP_08630058.1 glutathione S-transferase LigF[Bradyrhizobiaceae bacterium SG-6C] >gb|EGP07427.1|glutathione S-transferase [Bradyrhizobiaceae bacterium SG-6C] 377 378 AAG34806.1glutathione S-transferase GST 16 LigF [Glycine max] 379 380 AAQ02687.1tau class GST protein 3 [Oryza sativa LigF Indica Group] >gb|EAY79295.1|hypothetical protein OsI_34421 [Oryza sativa IndicaGroup] >emb|CAZ68078.1| glutathione S-transferase [Oryza sativa IndicaGroup] 381 382 ADV56298.1 Glutathione S-transferase domain LigF protein[Shewanella putrefaciens 200] 383 384 BAB70616.1 glutathioneS-transferase [Medicago LigF sativa] 385 386 BAJ94610.1 predictedprotein [Hordeum vulgare LigF subsp. vulgare] 387 388 CAN68934.1hypothetical protein VITISV_002763 LigF [Vitis vinifera] 389 390CBW26056.1 putative glutathione S-transferase LigF [Bacteriovoraxmarinus SJ] 391 392 EFW18159.1 glutathione S-transferase [CoccidioidesLigF posadasii str. Silveira] 393 394 EGF84337.1 hypothetical proteinLigF BATDEDRAFT_85058 [Batrachochytrium dendrobatidis JAM81] 395 396NP_001065124.1 Os10g0528400 [Oryza sativa Japonica LigFGroup] >gb|AAG32472.1|AF309379_1 putative glutathione S-transferaseOsGSTU3 [Oryza sativa Japonica Group] >gb|AAM12325.1|AC091680_26putative glutathione S-transferase [Oryza sativa JaponicaGroup] >gb|AAM94544.1|putative glutathione S- transferase [Oryza sativaJaponica Group] >gb|AAP54745.1|glutathione S- transferase GSTU6,putative, expressed [Oryza sativa JaponicaGroup] >dbj|BAF27038.1|Os10g0528400 [Oryza sativa JaponicaGroup] >gb|EAZ16756.1|hypothetical protein OsJ_32232 [Oryza sativaJaponica Group] 397 398 NP_191835.1 Glutathione S-transferase-likeprotein LigF [Arabidopsis thaliana] >emb|CAB83126.1|Glutathionetransferase III-like protein [Arabidopsisthaliana] >gb|AEE80388.1|Glutathione S-transferase-like protein[Arabidopsis thaliana] 399 400 NP_717190.1 glutathione S-transferasefamily protein LigF [Shewanella oneidensisMR-1] >gb|AAN54634.1|AE015603_8 glutathione S-transferase family protein[Shewanella oneidensis MR-1] 401 402 NP_769143.1 glutathioneS-transferase LigF [Bradyrhizobium japonicum USDA110] >dbj|BAC47768.1|glutathione S- transferase [Bradyrhizobiumjaponicum USDA 110] 403 404 NP_900642.1 glutathione transferase zeta 1LigF [Chromobacterium violaceum ATCC 12472] >gb|AAQ58646.1|probableglutathione transferase zeta 1 [Chromobacterium violaceum ATCC 12472]405 406 XP_001246353.1 glutathione S-transferase [Coccidioides LigFimmitis RS] 407 408 XP_002171087.1 PREDICTED: similar to glutathione S-LigF transferase [Hydra magnipapillata] 409 410 XP_002263386.1PREDICTED: hypothetical protein [Vitis LigFvinifera] >emb|CBI32223.3|unnamed protein product [Vitis vinifera] 411412 XP_002263424.1 PREDICTED: hypothetical protein [Vitis LigFvinifera] >emb|CBI32222.3|unnamed protein product [Vitis vinifera] 413414 XP_002272099.1 PREDICTED: hypothetical protein LigF isoform 2 [Vitisvinifera] 415 416 XP_002527848.1 glutathione s-transferase, putativeLigF [Ricinus communis] >gb|EEF34551.1| glutathione s-transferase,putative [Ricinus communis] 417 418 XP_002786341.1 GlutathioneS-transferase A, putative LigF [Perkinsus marinus ATCC50983] >gb|EER18137.1|Glutathione S- transferase A, putative [Perkinsusmarinus ATCC 50983] 419 420 XP_003066789.1 Glutathione S-transferase,putative LigF [Coccidioides posadasii C735 deltaSOWgp] >gb|EER24644.1|Glutathione S-transferase, putative [Coccidioidesposadasii C735 delta SOWgp] 421 422 XP_970577.1 PREDICTED: similar toganglioside- LigF induced differentiation-associated- protein 1[Tribolium castaneum] >gb|EFA00477.1|hypothetical proteinTcasGA2_TC003336 [Tribolium castaneum] 423 424 YP_001263559.1glutathione S-transferase domain- LigF containing protein [Sphingomonaswittichii RW1] >gb|ABQ69421.1| Glutathione S-transferase, N-terminaldomain [Sphingomonas wittichii RW1] 425 426 YP_001503032.1 glutathioneS-transferase domain- LigF containing protein [Shewanella pealeana ATCC700345] >gb|ABV88497.1| Glutathione S-transferase domain [Shewanellapealeana ATCC 700345] 427 428 YP_001516981.1 glutathione S-transferaseII LigF [Acaryochloris marina MBIC11017] >gb|ABW27665.1|glutathione S-transferase II [Acaryochloris marina MBIC11017] 429 430 YP_001615392.1glutathione S-transferase, [Sorangium LigF cellulosum ‘So ce56’] >emb|CAN94912.1|glutathione S- transferase, putative [Sorangiumcellulosum ‘So ce 56’] 431 432 YP_001685556.1 glutathione S-transferasedomain- LigF containing protein [Caulobacter sp.K31] >gb|ABZ73058.1|Glutathione S- transferase domain [Caulobacter sp.K31] 433 434 YP_001748054.1 glutathione S-transferase domain- LigFcontaining protein [Pseudomonas putida W619] >gb|ACA71685.1|GlutathioneS- transferase domain [Pseudomonas putida W619] 435 436 YP_001804371.1glutathione S-transferase [Cyanothece LigF sp. ATCC51142] >gb|ACB52305.1| glutathione S-transferase [Cyanothece sp. ATCC51142] 437 438 YP_002007283.1 glutathione s-transferase protein; gstaLigF protein [Cupriavidus taiwanensis LMG19424] >emb|CAQ71222.1|putative glutathione S-transferase protein; gstAprotein [Cupriavidus taiwanensis LMG 19424] 439 440 YP_002130812.1glutathione S-transferase LigF [Phenylobacterium zucineumHLK1] >gb|ACG78383.1|glutathione S- transferase [Phenylobacteriumzucineum HLK1] 441 442 YP_002220633.1 glutathione S-transferase domainLigF [Acidithiobacillus ferrooxidans ATCC 53993] >ref|YP_002426974.1|glutathione S-transferase [Acidithiobacillus ferrooxidans ATCC23270] >gb|ACH84426.1|Glutathione S- transferase domain[Acidithiobacillus ferrooxidans ATCC 53993] >gb|ACK78121.1|glutathioneS- transferase [Acidithiobacillus ferrooxidans ATCC 23270] 443 444YP_002482418.1 glutathione S-transferase domain- LigF containing protein[Cyanothece sp. PCC 7425] >gb|ACL44057.1|Glutathione S- transferasedomain protein [Cyanothece sp. PCC 7425] 445 446 YP_002543747.1glutathione S-transferase protein LigF [Agrobacterium radiobacterK84] >gb|ACM25821.1|glutathione S- transferase protein [Agrobacteriumradiobacter K84] 447 448 YP_002974739.1 glutathione S-transferase domainprotein LigF [Rhizobium leguminosarum bv. trifoliiWSM1325] >gb|ACS55200.1| Glutathione S-transferase domain protein[Rhizobium leguminosarum bv. trifolii WSM1325] 449 450 YP_004065207.1glutathione transferase LigF [Pseudoalteromonas sp.SM9913] >gb|ADT70298.1|glutathione transferase [Pseudoalteromonas sp.SM9913] 451 452 YP_004357179.1 glutathione S-transferase [PseudomonasLigF brassicacearum subsp. brassicacearumNFM421] >gb|AEA72175.1|putative glutathione S-transferase [Pseudomonasbrassicacearum subsp. brassicacearum NFM421] 453 454 YP_004680920.1glutathione S-transferase [Cupriavidus LigF necator N-1] >gb|AEI79688.1|glutathione S-transferase [Cupriavidus necator N-1] 455 456 YP_468810.1glutathione S-transferase [Rhizobium etli LigF CFN42] >gb|ABC90083.1|glutathione S- transferase protein [Rhizobium etliCFN 42] 457 458 YP_554040.1 glutathione S-transferase [Burkholderia LigFxenovorans LB400] >gb|ABE34690.1| Glutathione S-transferase[Burkholderia xenovorans LB400] 459 460 YP_612103.1 glutathioneS-transferase-like [Ruegeria LigF sp. TM1040] >gb|ABF62841.1|glutathione S-transferase-like protein [Ruegeria sp. TM1040] 461 462YP_735310.1 glutathione S-transferase domain- LigF containing protein[Shewanella sp. MR-4] >gb|ABI40253.1|Glutathione S- transferase,N-terminal domain protein [Shewanella sp. MR-4] 463 464 YP_747567.1glutathione S-transferase domain- LigF containing protein [Nitrosomonaseutropha C91] >gb|ABI59602.1| Glutathione S-transferase, C-terminaldomain [Nitrosomonas eutropha C91] 465 466 YP_757227.1maleylacetoacetate isomerase LigF [Maricaulis marisMCS10] >gb|ABI66289.1|maleylacetoacetate isomerase [Maricaulis marisMCS10] 467 468 YP_868399.1 glutathione S-transferase domain- LigFcontaining protein [Shewanella sp. ANA- 3] >gb|ABK46993.1|Glutathione S-transferase, N-terminal domain protein [Shewanella sp. ANA-3] 469 470YP_870498.1 glutathione S-transferase domain- LigF containing protein[Shewanella sp. ANA- 3] >gb|ABK49092.1|Glutathione S- transferase,N-terminal domain protein [Shewanella sp. ANA-3] 471 472 YP_957711.1glutathione S-transferase domain- LigF containing protein [Marinobacteraquaeolei VT8] >gb|ABM17524.1| Glutathione S-transferase, N-terminaldomain [Marinobacter aquaeolei VT8] 473 474 YP_957873.1 glutathioneS-transferase domain- LigF containing protein [Marinobacter aquaeoleiVT8] >gb|ABM17686.1| Glutathione S-transferase, N-terminal domain[Marinobacter aquaeolei VT8] 475 476 YP_960793.1 glutathioneS-transferase domain- LigF containing protein [Marinobacter aquaeoleiVT8] >gb|ABM20606.1| Glutathione S-transferase, N-terminal domain[Marinobacter aquaeolei VT8] 477 478 YP_963418.1 glutathioneS-transferase domain- LigF containing protein [Shewanella sp. W3-18-1] >gb|ABM24864.1|Glutathione S- transferase, N-terminal domain[Shewanella sp. W3-18-1] 479 480 ZP_01000028.1 glutathione S-transferasefamily protein LigF [Oceanicola batsensisHTCC2597] >gb|EAQ02499.1|glutathione S- transferase family protein[Oceanicola batsensis HTCC2597] 481 482 ZP_01459182.1 glutathioneS-transferase [Stigmatella LigF aurantiacaDW4/3-1] >ref|YP_003956548.1|glutathione s- transferase [Stigmatellaaurantiaca DW4/3-1] >gb|EAU70026.1|glutathione S-transferase[Stigmatella aurantiaca DW4/3-1] >gb|ADO74721.1|GlutathioneS-transferase [Stigmatella aurantiaca DW4/3-1] 483 484 ZP_02886014.1Glutathione S-transferase domain LigF [Burkholderia graminisC4D1M] >gb|EDT08402.1|Glutathione S- transferase domain [Burkholderiagraminis C4D1M] 485 486 ZP_04713937.1 Glutathione S-transferase[Alteromonas LigF macleodii ATCC 27126] 487 488 ZP_05075049.1Glutathione S-transferase, N-terminal LigF domain protein[Rhodobacterales bacterium HTCC2083] >gb|EDZ42709.1| GlutathioneS-transferase, N-terminal domain protein [Rhodobacteraceae bacteriumHTCC2083] 489 490 ZP_05101428.1 glutathione S-transferase protein LigF[Roseobacter sp. GAI101] >gb|EEB85730.1|glutathione S- transferaseprotein [Roseobacter sp. GAI101] 491 492 ZP_05124402.1 glutathioneS-transferase LigF [Rhodobacteraceae bacteriumKLH11] >gb|EEE39034.1|glutathione S- transferase [Rhodobacteraceaebacterium KLH11] 493 494 ZP_05926645.1 glutathione S-transferase [Vibriosp. LigF RC341] >gb|EEX64947.1|glutathione S- transferase [Vibrio sp.RC341] 495 496 ZP_06308936.1 Glutathione S-transferase-like protein LigF[Cylindrospermopsis raciborskii CS-505] >gb|EFA69058.1|Glutathione S-transferase-like protein [Cylindrospermopsis raciborskii CS-505] 497 498ZP_06838829.1 Glutathione S-transferase domain LigF protein[Burkholderia sp. Ch1-1] >gb|EFG73275.1|Glutathione S- transferasedomain protein [Burkholderia sp. Ch1-1] 499 500 ZP_08104209.1glutathione S-transferase III [Vibrio LigF sinaloensis DSM21326] >gb|EGA68654.1|glutathione S- transferase III [Vibrio sinaloensisDSM 21326] 501 502 ZP_08275708.1 Glutathione S-transferase LigF[Oxalobacteraceae bacterium IMCC9480] >gb|EGF30821.1| GlutathioneS-transferase [Oxalobacteraceae bacterium IMCC9480] 503 504ZP_08409706.1 glutathione S-transferase LigF [Pseudoalteromonashaloplanktis ANT/505] >gb|EGI73123.1|glutathione S$$ transferase[Pseudoalteromonas haloplanktis ANT/505] 505 506 ZP_08565123.1glutathione S-transferase [Shewanella LigF sp. HN-41] >gb|EGM70872.1|glutathione S-transferase [Shewanella sp. HN-41] 507 508 CAA12269.1 ORF3 [Sphingomonas sp. RW5] LigF 509 510 CAC94002.1 glutathione transferase[Triticum LigF aestivum] 511 512 NP_967294.1 maleylacetoacetateisomerase/ LigF glutathione S-transferase [Bdellovibrio bacteriovorusHD100] >emb|CAE77948.1|maleylacetoacetate isomerase/glutathioneS-transferase [Bdellovibrio bacteriovorusHD100] 513 514 P30347.1RecName: Full = Protein ligF LigF >dbj|BAA02031.1|beta-etherase[Sphingomonas paucimobilis] >prf||1914145A beta etherase 515 516XP_002964271.1 hypothetical protein LigF SELMODRAFT_142654 [Selaginellamoellendorffii] >gb|EFJ34604.1| hypothetical protein SELMODRAFT_142654[Selaginella moellendorffii] 517 518 YP_001021314.1 glutathioneS-transferase-like protein LigF [Methylibium petroleiphilumPM1] >gb|ABM95079.1|glutathione S- transferase-like protein [Methylibiumpetroleiphilum PM1] 519 520 YP_001862387.1 glutathione S-transferasedomain- LigF containing protein [Burkholderia phymatumSTM815] >gb|ACC75341.1| Glutathione S-transferase domain [Burkholderiaphymatum STM815] 521 522 YP_002130750.1 glutathione S-transferase LigF[Phenylobacterium zucineum HLK1] >gb|ACG78321.1|glutathione S-transferase [Phenylobacterium zucineum HLK1] 523 524 YP_002825255.1glutathione S-transferase [Sinorhizobium LigF frediiNGR234] >gb|ACP24502.1| glutathione S-transferase [Sinorhizobium frediiNGR234] 525 526 YP_003908670.1 glutathione S-transferase domain- LigFcontaining protein [Burkholderia sp. CCGE1003] >gb|ADN59379.1|Glutathione S-transferase domain protein [Burkholderia sp. CCGE1003] 527528 YP_004154430.1 glutathione s-transferase domain- LigF containingprotein [Variovorax paradoxus EPS] >gb|ADU36319.1|Glutathione S-transferase domain [Variovorax paradoxus EPS] 529 530 YP_004229981.1glutathione S-transferase domain- LigF containing protein [Burkholderiasp. CCGE1001] >gb|ADX56921.1| Glutathione S-transferase domain protein[Burkholderia sp. CCGE1001] 531 532 YP_004302768.1 glutathioneS-transferase, N-terminal LigF domain protein [Polymorphum gilvumSL003B-26A1] >gb|ADZ69468.1| Glutathione S-transferase, N-terminaldomain protein [Polymorphum gilvum SL003B-26A1] 533 534 YP_004533892.1glutathione S-transferase-like protein LigF [Novosphingobium sp.PP1Y] >emb|CCA92074.1|glutathione S- transferase-like [Novosphingobiumsp. PP1Y] 535 536 YP_004533893.1 glutathione S-transferase-like proteinLigF [Novosphingobium sp. PP1Y] >emb|CCA92075.1|glutathione S-transferase-like [Novosphingobium sp. PP1Y] 537 538 YP_004533905.1glutathione S-transferase-like protein LigF [Novosphingobium sp.PP1Y] >emb|CCA92087.1|glutathione S- transferase-like [Novosphingobiumsp. PP1Y] 539 540 YP_497364.1 glutathione S-transferase-like proteinLigF [Novosphingobium aromaticivorans DSM12444] >gb|ABD26530.1|glutathione S- transferase-like protein[Novosphingobium aromaticivorans DSM 12444] 541 542 YP_498135.1glutathione S-transferase-like protein LigF [Novosphingobiumaromaticivorans DSM 12444] >gb|ABD27301.1|glutathione S-transferase-like protein [Novosphingobium aromaticivorans DSM 12444] 543544 YP_498142.1 glutathione S-transferase-like protein LigF[Novosphingobium aromaticivorans DSM 12444] >gb|ABD27308.1|glutathioneS- transferase-like protein [Novosphingobium aromaticivorans DSM 12444]545 546 YP_498143.1 glutathione S-transferase-like protein LigF[Novosphingobium aromaticivorans DSM 12444] >gb|ABD27309.1|glutathioneS- transferase-like protein [Novosphingobium aromaticivorans DSM 12444]547 548 ZP_00952372.1 maleylacetoacetate isomerase LigF [Oceanicaulisalexandrii HTCC2633] >gb|EAP91525.1|maleylacetoacetate isomerase[Oceanicaulis alexandrii HTCC2633] 549 550 ZP_00959702.1 glutathioneS-transferase, putative LigF [Roseovarius nubinhibensISM] >gb|EAP78164.1|glutathione S- transferase, putative [Roseovariusnubinhibens ISM] 551 552 ZP_01034543.1 glutathione S-transferase,putative LigF [Roseovarius sp. 217] >gb|EAQ27224.1| glutathioneS-transferase, putative [Roseovarius sp. 217] 553 554 ZP_01057917.1glutathione S-transferase, putative LigF [Roseobacter sp.MED193] >gb|EAQ44057.1|glutathione S- transferase, putative [Roseobactersp. MED193] 555 556 ZP_01223510.1 glutathione S-transferase [marine LigFgamma proteobacterium HTCC2207] >gb|EAS48069.1|glutathione S-transferase [marine gamma proteobacterium HTCC2207] 557 558ZP_01753989.1 glutathione S-transferase, putative LigF [Roseobacter sp.SK209-2-6] >gb|EBA17470.1|glutathione S- transferase, putative[Roseobacter sp. SK209-2-6] 559 560 ZP_02146800.1 glutathioneS-transferase-like protein LigF [Phaeobacter gallaeciensisBS107] >gb|EDQ11817.1|glutathione S- transferase-like protein[Phaeobacter gallaeciensis BS107] 561 562 ZP_02150992.1 glutathioneS-transferase, putative LigF [Phaeobacter gallaeciensis2.10] >gb|EDQ07480.1|glutathione S- transferase, putative [Phaeobactergallaeciensis 2.10] 563 564 ZP_05073592.1 glutathione S-transferase 2LigF [Rhodobacterales bacterium HTCC2083] >gb|EDZ41252.1|glutathione S-transferase 2 [Rhodobacteraceae bacterium HTCC2083] 565 566ZP_05077451.1 glutathione S-transferase LigF [Rhodobacterales bacteriumY4I] >gb|EDZ45430.1|glutathione S- transferase [Rhodobacteralesbacterium Y4I] 567 568 ZP_05087035.1 Glutathione S-transferase,N-terminal LigF domain protein [Pseudovibrio sp.JE062] >gb|EEA92555.1|Glutathione S- transferase, N-terminal domainprotein [Pseudovibrio sp. JE062] 569 570 ZP_05089424.1 glutathioneS-transferase [Ruegeria sp. LigF R11] >gb|EEB71116.1|glutathione S-transferase [Ruegeria sp. R11] 571 572 ZP_05126316.1 protein LigF [gammaproteobacterium LigF NOR5-3] >gb|EED32863.1|protein LigF [gammaproteobacterium NOR5-3] 573 574 ZP_05126823.1 maleylacetoacetateisomerase [gamma LigF proteobacteriumNOR5-3] >gb|EED33370.1|maleylacetoacetate isomerase [gammaproteobacterium NOR5-3] 575 576 ZP_05741946.1 glutathione S-transferase[Silicibacter sp. LigF TrichCH4B] >gb|EEW58747.1| glutathioneS-transferase [Silicibacter sp. TrichCH4B]

TABLE 18 PROTEIN GENE GENBANK SEQ ID SEQ ID ACCESSION NO: NO: NO:DESCRIPTION: TYPE 577 578 BAA77216.1 glutathione S-transferase homologLigG [Sphingomonas paucimobilis] 579 580 YP_004533907.

glutathione S-transferase family protein LigG [Novosphingobium sp.PP1Y] >emb|CCA92089.1|glutathione S- transferase family protein 581 582YP_314808.1 glutathione S-transferase family protein LigG [Thiobacillusdenitrificans ATCC 25259] >gb|AAZ97003.1|glutathione S- transferasefamily protein [Thiobacillus 583 584 YP_167289.1 glutathioneS-transferase family protein LigG [Ruegeria pomeroyiDSS-3] >gb|AAV95330.1|glutathione S- transferase family protein[Ruegeria 585 586 ZP_01011943.1 glutathione S-transferase family proteinLigG [Maritimibacter alkaliphilus HTCC2654] >gb|EAQ14262.1|glutathioneS- transferase family protein 587 588 YP_002540613.

glutathione S-transferase protein LigG [Agrobacterium radiobacterK84] >gb|ACM29018.1|glutathione S- 589 590 CAJ81793.1 Novel glutathioneS-transferase omega LigG protein [Xenopus (Silurana) tropicalis] 591 592NP_001005086.

glutathione S-transferase omega 2 LigG [Xenopus (Silurana)tropicalis] >gb|AAH77010.1|MGC89704 protein 593 594 XP_624501.1PREDICTED: glutathione S-transferase LigG omega-1 [Apis mellifera] 595596 XP_002029736.

GM24932 [Drosophila sechellia] LigG >gb|EDW40722.1|GM24932 [Drosophila597 598 NP_001002621.

hypothetical protein LOC436894 [Danio LigG rerio ] >gb|AAH75965.1|Zgc:92254 [Danio 599 600 XP_002431486.

predicted protein [Pediculus humanus LigG corporis] >gb|EEB18748.1|predicted protein [Pediculus humanus corporis] 601 602ADD18952.1 glutathione S-transferase [Glossina LigG morsitans morsitans]603 604 XP_002093444.

GE21298 [Drosophila yakuba] LigG >gb|EDW93156.1|GE21298 [Drosophila 605606 XP_002068563.

GK20540 [Drosophila willistoni] LigG >gb|EDW79549.1|GK20540 [Drosophila607 608 NP_001165912.

glutathione S-transferase O1 [Nasonia LigG 609 610 CAM34501.1 putativeglutathione S-transferase LigG [Cotesia congregata] 611 612 XP_421747.1PREDICTED: similar to glutathione-S- LigG transferase homolog isoform 2[Gallus 613 614 XP_002135069.

GA23449 [Drosophila pseudoobscura LigG pseudoobscura ] >gb|EDY73696.1|GA23449 [Drosophila pseudoobscura 615 616 NP_034492.1 glutathioneS-transferase omega-1 [Mus LigG musculus] >sp|O09131.2|GSTO1_MOUSERecName: Full = Glutathione S-transferase omega-1; Short = GSTO-1;AltName: Full = p28 >gb|AAB70110.1|glutathione-S- transferase homolog[Mus musculus] >dbj|BAC25667.1|unnamed protein product [Musmusculus] >gb|AAH85165.1|Glutathione S- transferase omega 1 [Musmusculus] >dbj|BAE27469.1|unnamed protein product [Mus musculus]

617 618 ZP_03524422.1 glutathione S-transferase domain- LigG containingprotein [Rhizobium etli GR56] 619 620 NP_729388.1 CG6673, isoform A[Drosophila LigG melanogaster ] >gb|AAF50404.2|CG6673, isoform A[Drosophila melanogaster] >gb|ACZ02426.1|glutathione S- 621 622ZP_08179398.1 glutathione S-transferase [Xanthomonas LigG vesicatoriaATCC 35937] >gb|EGD08414.1|glutathione S- 623 624 XP_003218563.

PREDICTED: glutathione S-transferase LigG omega-1-like isoform 1 [Anolis625 626 ABC86304.1 IP16242p [Drosophila melanogaster] LigG 627 628XP_002026470.

GL15567 [Drosophila persimilis] LigG >gb|EDW33419.1|GL15567 [Drosophila629 630 NP_001108461.

glutathione S-transferase omega 4 LigG [Bombyx mori ] >gb|ABY66601.1|glutathione S-transferase 13 [Bombyx 631 632 NP_999215.1 glutathioneS-transferase omega-1 [Sus LigG scrofa ] >ref|XP_001929519.1| PREDICTED:glutathione S-transferase omega-1-like [Susscrofa] >sp|Q9N1F5.2|GSTO1_PIG RecName: Full = Glutathione S-transferaseomega-1; Short = GSTO-1; AltName: Full = Glutathione-dependentdehydroascorbate reductase 633 634 NP_001007373.

hypothetical protein LOC492500 [Danio LigG rerio ] >gb|AAH85467.1|Zgc:101897 [Danio rerio ] >gb|AAI65433.1|Zgc: 101897 635 636 YP_001566654.

glutathione S-transferase domain- LigG containing protein [Delftiaacidovorans SPH-1] >gb|ABX38269.1|Glutathione S- transferase domain[Delftia acidovorans 637 638 ADY80021.1 omega class glutathioneS-transferase LigG [Oplegnathus fasciatus] 639 640 YP_001329158.

glutathione S-transferase domain- LigG containing protein [Sinorhizobiummedicae WSM419] >gb|ABR62323.1| Glutathione S-transferase domain 641 642NP_001084924.

hypothetical protein LOC431979 LigG [Xenopus laevis ] >gb|AAH70673.1|MGC82327 protein [Xenopus laevis] 643 644 XP_003396907.

PREDICTED: glutathione S-transferase LigG omega-1-like [Bombusterrestris] 645 646 XP_001368758.

PREDICTED: glutathione S-transferase LigG omega-1-like isoform 1[Monodelphis 647 648 XP_001983981.

GH16193 [Drosophila grimshawi] LigG >gb|EDV96329.1|GH16193 [Drosophila649 650 ADK66966.1 glutathione s-transferase [Chironomus LigG 651 652XP_001232808.

PREDICTED: similar to glutathione-S- LigG transferase homolog isoform 1[Gallus 653 654 XP_002068565.

GK20354 [Drosophila willistoni] LigG >gb|EDW79551.1|GK20354 [Drosophila655 656 YP_001611239.

hypothetical protein sce0602 [Sorangium LigG cellulosum ‘So ce56’] >emb|CAN90759.1| gst2 [Sorangium cellulosum ‘So ce 56’] 657 658XP_001499427.

PREDICTED: glutathione S-transferase LigG omega-1-like isoform 1 [Equuscaballus] 659 660 NP_384409.1 putative glutathione S-transferase proteinLigG [Sinorhizobium meliloti 1021] >ref|YP_004550950.1|glutathione S-transferase domain-containing protein [Sinorhizobium melilotiAK83] >emb|CAC41740.1|Putative glutathione S-transferase [Sinorhizobiummeliloti 1021] >gb|AEG06303.1|Glutathione S- transferase domain protein[Sinorhizobium meliloti BL225C] >gb|AEG55336.1|Glutathione S-

661 662 CAG05035.1 unnamed protein product [Tetraodon LigG 663 664ZP_01365353.1 hypothetical protein PaerPA_01002475 LigG [Pseudomonasaeruginosa PACS2] >ref|YP_002440902.1| maleylacetoacetate isomerase[Pseudomonas aeruginosa LESB58] >ref|ZP_04928412.1|maleylacetoacetateisomerase [Pseudomonas aeruginosa C3719] >gb|EAZ52531.1|maleylacetoacetate isomerase [Pseudomonas aeruginosaC3719] >emb|CAW28043.1|maleylacetoacetate isomerase [Pseudomonasaeruginosa

665 666 YP_001348642.

maleylacetoacetate isomerase LigG [Pseudomonas aeruginosaPA7] >gb|ABR84080.1|maleylacetoacetate isomerase [Pseudomonas aeruginosa667 668 ZP_04933765.1 maleylacetoacetate isomerase LigG [Pseudomonasaeruginosa 2192] >gb|EAZ57884.1|maleylacetoacetate isomerase[Pseudomonas aeruginosa 669 670 NP_250697.1 maleylacetoacetate isomeraseLigG [Pseudomonas aeruginosa PAO1] >sp|P57109.1|MAAI_PSEAE RecName: Full= Maleylacetoacetate isomerase; Short = MAAI >gb|AAG05395.1|AE004627_3671 672 EFN59352.1 hypothetical protein LigG CHLNCDRAFT_137800[Chlorella 673 674 YP_002945584.

glutathione S-transferase domain- LigG containing protein [Variovoraxparadoxus S110] >gb|ACS20318.1|Glutathione S- transferase domain protein[Variovorax 675 676 XP_002197460.

PREDICTED: glutathione S-transferase LigG omega 1 [Taeniopygia guttata]677 678 XP_001971643.

GG15075 [Drosophila erecta] LigG >gb|EDV50669.1|GG15075 [Drosophila 679680 NP_001155757.

glutathione S-transferase omega-1-like LigG [Acyrthosiphon pisum] >dbj|BAH71013.1| ACYPI008340 [Acyrthosiphon pisum] 681 682XP_002026468.

GL15565 [Drosophila persimilis] LigG >gb|EDW33417.1|GL15565 [Drosophila683 684 XP_001353820.

GA19760 [Drosophila pseudoobscura LigG pseudoobscura ] >gb|EAL29555.1|GA19760 [Drosophila pseudoobscura 685 686 YP_791232.1 maleylacetoacetateisomerase LigG [Pseudomonas aeruginosaUCBPP-PA14] >gb|ABJ11194.1|maleylacetoacetate isomerase [Pseudomonasaeruginosa 687 688 ZP_06879058.1 maleylacetoacetate isomerase LigG[Pseudomonas aeruginosa PAb1] >ref|ZP_07797003.1|maleylacetoacetateisomerase [Pseudomonas aeruginosa 39016] >gb|EFQ42099.1|maleylacetoacetate isomerase [Pseudomonas aeruginosa39016] >gb|EGM14661.1|maleylacetoacetate 689 690 EFZ22366.1 hypotheticalprotein SINV_14968 LigG 691 692 ZP_03527925.1 Glutathione S-transferasedomain LigG [Rhizobium etli CIAT 894] 693 694 ABD77536.1 hypotheticalprotein [Ictalurus punctatus] LigG 695 696 XP_002756473.

PREDICTED: glutathione S-transferase LigG omega-1-like [Callithrixjacchus] 697 698 XP_001636996.

predicted protein [Nematostella vectensis] LigG >gb|EDO44933.1|predictedprotein [Nematostella vectensis] 699 700 YP_467831.1 glutathioneS-transferase [Rhizobium etli LigG CFN 42] >gb|ABC89104.1|glutathione S-transferase protein [Rhizobium etli CFN 701 702 NP_103005.1glutathione-S-transferase [Mesorhizobium LigG lotiMAFF303099] >dbj|BAB48791.1| glutathione-S-transferase [Mesorhizobium703 704 ADY47623.1 Glutathione transferase omega-1 [Ascaris LigG 705 706BAG36430.1 unnamed protein product [Homo sapiens] LigG 707 708XP_002718774.

PREDICTED: glutathione-S-transferase LigG omega 1 [Oryctolaguscuniculus] 709 710 3LFL_A Chain A, Crystal Structure Of Human LigGGlutathione Transferase Omega 1, Delta 155 >pdb|3LFL|B Chain B, CrystalStructure Of Human Glutathione Transferase Omega 1, Delta155 >pdb|3LFL|C Chain C, Crystal Structure 711 712 XP_002805857.

PREDICTED: glutathione S-transferase LigG omega-1-like [Macacamulatta] >gb|ABO21635.1|glutathione S- 713 714 NP_001007603.

glutathione S-transferase omega-1 LigG [Rattus norvegicus] >gb|AAH79363.1| Glutathione S-transferase omega 1 [Rattus norvegicus] >gb|EDL94393.1| glutathione S-transferase omega 1, 715 716 XP_535007.1PREDICTED: similar to glutathione-S- LigG transferase omega 1 isoform 1[Canis 717 718 NP_004823.1 glutathione S-transferase omega-1 LigGisoform 1 [Homo sapiens] >sp|P78417.2|GSTO1_HUMAN RecName: Full =Glutathione S-transferase omega-1; Short = GSTO-1 >pdb|1EEM|A Chain A,Glutathione Transferase From Homo Sapiens >gb|AAF73376.1|AF212303_1glutathione transferase omega [Homosapiens] >gb|AAB70109.1|glutathione-S- transferase homolog [Homosapiens] >gb|AAH00127.1|Glutathione S- transferase omega 1 [Homosapiens] >gb|AAV68046.1|glutathione S- transferase omega 1-1 [Homosapiens]

719 720 XP_002758417.

PREDICTED: glutathione S-transferase LigG omega-1-like [Callithrixjacchus] 721 722 XP_003218564.

PREDICTED: glutathione S-transferase LigG omega-1-like isoform 2 [Anolis723 724 EFN62827.1 Glutathione transferase omega-1 LigG [Camponotusfloridanus] 725 726 XP_508020.3 PREDICTED: glutathione S-transferaseLigG omega-1 isoform 3 [Pan troglodytes] 727 728 CAD97673.1 hypotheticalprotein [Homo sapiens] LigG 729 730 BAJ20927.1 glutathione S-transferaseomega 1 LigG [synthetic construct] 731 732 ACR43779.1 glutathioneS-transferase [Chironomus LigG 733 734 Q9Z339.2 RecName: Full =Glutathione S-transferase LigG omega-1; Short = GSTO-1; AltName: Full =Glutathione-dependent dehydroascorbatereductase >gb|ACI32122.1|glutathione S- 735 736 XP_001956909.

GF10159 [Drosophila ananassae] LigG >gb|EDV39715.1|GF10159 [Drosophila737 738 XP_001742278.

hypothetical protein [Monosiga brevicollis LigGMX1] >gb|EDQ92516.1|predicted protein [Monosiga brevicollis MX1] 739 740XP_002821176.

PREDICTED: glutathione S-transferase LigG omega-1-like [Pongo abelii]741 742 XP_003255483.

PREDICTED: glutathione S-transferase LigG omega-1-like isoform 1[Nomascus 743 744 YP_325490.1 glutathione S-transferase-like proteinLigG [Anabaena variabilis ATCC 29413] >gb|ABA24595.1|Glutathione S-transferase-like protein [Anabaena 745 746 XP_003208190.

PREDICTED: glutathione S-transferase LigG omega-1-like [Meleagrisgallopavo] 747 748 XP_002068562.

GK20539 [Drosophila willistoni] LigG >gb|EDW79548.1|GK20539 [Drosophila749 750 XP_001956911.

GF10161 [Drosophila ananassae] LigG >gb|EDV39717.1|GF10161 [Drosophila751 752 ABV24048.1 gluthathione S-transferase omega LigG [Takifuguobscurus] 753 754 ZP_05086262.1 putative glutathione S-transferaseprotein LigG [Pseudovibrio sp. JE062] >gb|EEA93528.1|putativeglutathione S- transferase protein [Pseudovibrio sp. 755 756 AAI28951.1LOC100037104 protein [Xenopus laevis] LigG 757 758 XP_001956910.

GF10160 [Drosophila ananassae] LigG >gb|EDV39716.1|GF10160 [Drosophila759 760 NP_001099052.

glutathione S-transferase omega 2 LigG [Xenopus laevis ] >gb|AAI53758.1|LOC100037104 protein [Xenopus laevis] 761 762 ZP_03503214.1 GlutathioneS-transferase domain LigG [Rhizobium etli Kim 5] 763 764 XP_002046961.

GJ12198 [Drosophila virilis] LigG >gb|EDW69303.1|GJ12198 [Drosophila 765766 XP_001956912.

GF24331 [Drosophila ananassae] LigG >gb|EDV39718.1|GF24331 [Drosophila767 768 XP_001368790.

PREDICTED: glutathione S-transferase LigG omega-1-like isoform 1[Monodelphis 769 770 ZP_06308936.1 Glutathione S-transferase-likeprotein LigG [Cylindrospermopsis raciborskiiCS-505] >gb|EFA69058.1|Glutathione S- transferase-like protein 771 772ABJ15788.1 glutathione S-transferase omega 1 LigG [Bombyx mandarina] >dbj|BAF91356.1| omega-class glutathione S-transferase 773 774NP_001037406.

glutathione S-transferase omega 2 LigG [Bombyx mori ] >gb|ABC79689.1|glutathione S-transferase 6 [Bombyx mori] 775 776 NP_001040131.

glutathione S-transferase omega 1 LigG [Bombyx mori ] >gb|ABD36128.1|glutathione S-transferase omega 1

indicates data missing or illegible when filed

TABLE 19 PROTEIN GENE SEQ ID SEQ ID GENBANK NO: NO: ACCESSION NO:DESCRIPTION: TYPE 777 778 Q01198.1 RecName: Full = C alpha-dehydrogenaseLigD >dbj|BAA02030.1|C alpha-dehydrogenase [Sphingomonaspaucimobilis] >dbj|BAA01953.1|C alpha-dehydrogenase [Sphingomonaspaucimobilis] >gb|AAC60455.1|C alpha-dehydrogenase [Sphingomonaspaucimobilis] 779 780 YP_495487.1 short-chain dehydrogenase/reductaseSDR LigD [Novosphingobium aromaticivorans DSM12444] >gb|ABD24653.1|short-chain dehydrogenase/reductase SDR[Novosphingobium aromaticivorans DSM 12444] 781 782 YP_004533898.1short-chain dehydrogenase/reductase SDR LigD [Novosphingobium sp.PP1Y] >emb|CCA92080.1|short-chain dehydrogenase/reductase SDR[Novosphingobium sp. PP1Y] 783 784 BAH56687.1 Calpha-dehydrogenase[Sphingobium sp. LigD SYK-6] 785 786 YP_004533921.1 short-chaindehydrogenase/reductase SDR LigD [Novosphingobium sp.PP1Y] >emb|CCA92103.1|short-chain dehydrogenase/reductase SDR[Novosphingobium sp. PP1Y] 787 788 YP_496072.1 short-chaindehydrogenase/reductase SDR LigD [Novosphingobium aromaticivorans DSM12444] >gb|ABD25238.1|short-chain dehydrogenase/reductase SDR[Novosphingobium aromaticivorans DSM 12444] 789 790 3IOY_A Chain A,Structure Of Putative Short-Chain LigD Dehydrogenase (Saro_0793) FromNovosphingobium Aromaticivorans >pdb|3IOY|B Chain B, Structure OfPutative Short-Chain Dehydrogenase (Saro_0793) From NovosphingobiumAromaticivorans 791 792 YP_496073.1 short-chain dehydrogenase/reductaseSDR LigD [Novosphingobium aromaticivorans DSM12444] >gb|ABD25239.1|short-chain dehydrogenase/reductase SDR[Novosphingobium aromaticivorans DSM 12444] 793 794 BAH56683.1Calpha-dehydrogenase [Sphingobium sp. LigD SYK-6] 795 796 YP_004533920.1short-chain dehydrogenase/reductase SDR LigD [Novosphingobium sp.PP1Y] >emb|CCA92102.1|short-chain dehydrogenase/reductase SDR[Novosphingobium sp. PP1Y] 797 798 YP_003592832.1 short-chaindehydrogenase/reductase SDR LigD [Caulobacter segnis ATCC21756] >gb|ADG10214.1|short-chain dehydrogenase/reductase SDR[Caulobacter segnis ATCC 21756] 799 800 YP_495984.1 short-chaindehydrogenase/reductase SDR LigD [Novosphingobium aromaticivorans DSM12444] >gb|ABD25150.1|short-chain dehydrogenase/reductase SDR[Novosphingobium aromaticivorans DSM 12444] 801 802 YP_497149.1short-chain dehydrogenase/reductase SDR LigD [Novosphingobiumaromaticivorans DSM 12444] >gb|ABD26315.1|short-chaindehydrogenase/reductase SDR [Novosphingobium aromaticivorans DSM 12444]803 804 YP_003592830.1 short-chain dehydrogenase/reductase SDR LigD[Caulobacter segnis ATCC 21756] >gb|ADG10212.1|short-chaindehydrogenase/reductase SDR [Caulobacter segnis ATCC 21756] 805 806YP_001260886.1 short-chain dehydrogenase/reductase SDR LigD[Sphingomonas wittichii RW1] >gb|ABQ66748.1|short-chaindehydrogenase/reductase SDR [Sphingomonas wittichii RW1] 807 808YP_001413979.1 short-chain dehydrogenase/reductase SDR LigD[Parvibaculum lavamentivorans DS-1] >gb|ABS64322.1|short-chaindehydrogenase/reductase SDR [Parvibaculum lavamentivorans DS-1] 809 810YP_001412300.1 short-chain dehydrogenase/reductase SDR LigD[Parvibaculum lavamentivorans DS-1] >gb|ABS62643.1|short-chaindehydrogenase/reductase SDR [Parvibaculum lavamentivorans DS-1] 811 812YP_001412299.1 short-chain dehydrogenase/reductase SDR LigD[Parvibaculum lavamentivorans DS-1] >gb|ABS62642.1|short-chaindehydrogenase/reductase SDR [Parvibaculum lavamentivorans DS-1] 813 814BAH56685.1 Calpha-dehydrogenase [Sphingobium sp. LigD SYK-6] 815 816NP_959644.1 short chain dehydrogenase [Mycobacterium LigD avium subsp.paratuberculosis K-10] >ref|YP_880159.1|short chain dehydrogenase[Mycobacterium avium 104] >ref|ZP_05215302.1|short chain dehydrogenase[Mycobacterium avium subsp. avium ATCC25291] >gb|AAS03027.1|hypothetical protein MAP_0710c [Mycobacteriumavium subsp. paratuberculosis K-10] >gb|ABK67661.1| short chaindehydrogenase [Mycobacterium avium 104] >gb|EGO40035.1|short-chainalcohol dehydrogenase [Mycobacterium avium subsp. paratuberculosis S397]817 818 ZP_08717023.1 short chain dehydrogenase [Mycobacterium LigDcolombiense CECT 3035] >gb|EGT85268.1| short chain dehydrogenase[Mycobacterium colombiense CECT 3035] 819 820 ZP_05127447.1oxidoreductase, short chain LigD dehydrogenase/reductase family protein[gamma proteobacterium NOR5-3] >gb|EED33994.1|oxidoreductase, shortchain dehydrogenase/reductase family protein [gamma proteobacteriumNOR5-3] 821 822 YP_004555419.1 Estradiol 17-beta-dehydrogenase LigD[Sphingobium chlorophenolicum L-1] >gb|AEG50913.1|Estradiol 17-beta-dehydrogenase [Sphingobium chlorophenolicum L-1] 823 824 YP_004230838.1short-chain dehydrogenase/reductase SDR LigD [Burkholderia sp.CCGE1001] >gb|ADX57778.1|short-chain dehydrogenase/reductase SDR[Burkholderia sp. CCGE1001] 825 826 YP_004284589.1 putativeoxidoreductase [Acidiphilium LigD multivorum AIU301] >dbj|BAJ81707.1|putative oxidoreductase [Acidiphilium multivorum AIU301] 827 828YP_001235233.1 hypothetical protein Acry_2115 [Acidiphilium LigD cryptumJF-5] >gb|ABQ31314.1|short-chain dehydrogenase/reductase SDR[Acidiphilium cryptum JF-5] 829 830 ZP_01617820.1 hypothetical proteinGP2143_09415 [marine LigD gamma proteobacteriumHTCC2143] >gb|EAW30413.1|hypothetical protein GP2143_09415 [marine gammaproteobacterium HTCC2143] 831 832 ZP_08629833.1 short-chaindehydrogenase/reductase LigD [Bradyrhizobiaceae bacteriumSG-6C] >gb|EGP07476.1|short-chain dehydrogenase/reductase[Bradyrhizobiaceae bacterium SG-6C] 833 834 YP_001853014.1 short-chaintype dehydrogenase/reductase LigD [Mycobacterium marinumM] >gb|ACC43159.1|short-chain type dehydrogenase/reductase[Mycobacterium marinum M] 835 836 YP_004754457.1 short-chaindehydrogenase/reductase SDR LigD [Collimonas fungivoransTer331] >gb|AEK63634.1|short-chain dehydrogenase/reductase SDR[Collimonas fungivorans Ter331] 837 838 ZP_05129129.1 short-chaindehydrogenase/reductase SDR LigD [gamma proteobacteriumNOR5-3] >gb|EED30944.1|short-chain dehydrogenase/reductase SDR [gammaproteobacterium NOR5-3] 839 840 ZP_05223648.1 short chain dehydrogenase[Mycobacterium LigD intracellulare ATCC 13950] 841 842 YP_004555383.1short-chain dehydrogenase/reductase SDR LigD [Sphingobiumchlorophenolicum L-1] >gb|AEG50877.1|short-chain dehydrogenase/reductaseSDR [Sphingobium chlorophenolicum L-1] 843 844 YP_976997.1 short chaindehydrogenase [Mycobacterium LigD bovis BCG str. Pasteur1173P2] >ref|YP_002643932.1|short-chain dehydrogenase [Mycobacteriumbovis BCG str. Tokyo 172] >ref|ZP_06432004.1|short- chain typedehydrogenase/reductase [Mycobacterium tuberculosisT46] >ref|ZP_06449040.1|short-chain type dehydrogenase/reductase[Mycobacterium tuberculosis T17] >ref|ZP_06453700.1|short- chain typedehydrogenase/reductase [Mycobacterium tuberculosisK85] >ref|ZP_06508748.1|short-chain type dehydrogenase/reductase[Mycobacterium tuberculosis T92] >ref|ZP_06512283.1|short chaindehydrogenase [Mycobacterium tuberculosis EAS054] >ref|YP_004722558.1|short-chain type dehydrogenase/reductase [Mycobacterium africanumGM041182] >emb|CAL70889.1|Putative short-chain typedehydrogenase/reductase [Mycobacterium bovis BCG str. Pasteur1173P2] >dbj|BAH25164.1|short-chain dehydrogenase [Mycobacterium bovisBCG str. Tokyo 172] >gb|EFD12419.1|short- chain typedehydrogenase/reductase [Mycobacterium tuberculosisT46] >gb|EFD42482.1|short-chain type dehydrogenase/reductase[Mycobacterium tuberculosis K85] >gb|EFD46215.1|short- 845 846ZP_01101659.1 Short-chain dehydrogenase/reductase SDR LigD[Congregibacter litoralis KT71] >gb|EAQ98875.1|Short-chaindehydrogenase/reductase SDR [Congregibacter litoralis KT71] 847 848ZP_01615364.1 short chain dehydrogenase [marine gamma LigDproteobacterium HTCC2143] >gb|EAW32447.1|short chain dehydrogenase[marine gamma proteobacterium HTCC2143] 849 850 ZP_06436160.1short-chain type dehydrogenase/reductase LigD [Mycobacteriumtuberculosis CPHL_A] >gb|EFD16575.1|short-chain typedehydrogenase/reductase [Mycobacterium tuberculosis CPHL_A] 851 852NP_854532.1 short chain dehydrogenase [Mycobacterium LigD bovisAF2122/97] >emb|CAD93736.1| PUTATIVE SHORT-CHAIN TYPEDEHYDROGENASE/REDUCTASE [Mycobacterium bovis AF2122/97] 853 854YP_004744317.1 putative short-chain type LigD dehydrogenase/reductase[Mycobacterium canettii CIPT 140010059] >emb|CCC43191.1|putativeshort-chain type dehydrogenase/reductase [Mycobacterium canettii CIPT140010059] 855 856 YP_003947586.1 short-chain dehydrogenase/reductasesdr LigD [Paenibacillus polymyxa SC2] >gb|ADO57345.1|Short-chaindehydrogenase/reductase SDR [Paenibacillus polymyxa SC2] 857 858YP_003951191.1 short-chain dehydrogenase/reductase LigD [Stigmatellaaurantiaca DW4/3-1] >gb|ADO69364.1|Short-chain dehydrogenase/reductaseSDR [Stigmatella aurantiaca DW4/3-1] 859 860 YP_583994.1 hypotheticalprotein Rmet_1846 LigD [Cupriavidus metalliduransCH34] >gb|ABF08725.1|conserved hypothetical protein [Cupriavidusmetallidurans CH34] 861 862 NP_215366.1 short chain dehydrogenase[Mycobacterium LigD tuberculosis H37Rv] >ref|YP_001282151.1| short chaindehydrogenase [Mycobacterium tuberculosis H37Ra] >ref|YP_001286813.1|short chain dehydrogenase [Mycobacterium tuberculosisF11] >ref|ZP_02549252.1|short chain dehydrogenase [Mycobacteriumtuberculosis H37Ra] >ref|YP_003033128.1| short-chain typedehydrogenase/reductase [Mycobacterium tuberculosis KZN1435] >ref|ZP_04924487.1|hypothetical protein TBCG_00842 [Mycobacteriumtuberculosis C] >ref|ZP_04979832.1|hypothetical short- chain typedehydrogenase/reductase [Mycobacterium tuberculosis str.Haarlem] >ref|ZP_05140274.1|short chain dehydrogenase [Mycobacteriumtuberculosis ‘98-R604 INH-RIF-EM’] >ref|ZP_06444578.1|short-chain typedehydrogenase/reductase [Mycobacterium tuberculosis KZN605] >ref|ZP_06503955.1| short chain dehydrogenase [Mycobacteriumtuberculosis 02_1987] >ref|ZP_06516315.1| short chain dehydrogenase[Mycobacterium tuberculosis T85] >ref|ZP_06520361.1|short- chain typedehydrogenase/reductase [Mycobacterium tuberculosis GM1503] >ref|ZP_06802023.1|short chain dehydrogenase [Mycobacteriumtuberculosis 210] >ref|ZP_06951148.1|short chain 863 864 YP_904525.1short chain dehydrogenase [Mycobacterium LigD ulceransAgy99] >gb|ABL03054.1|short- chain type dehydrogenase/reductase[Mycobacterium ulcerans Agy99] 865 866 ZP_06851131.1 short-chaindehydrogenase/reductase family LigD oxidoreductase [Mycobacteriumparascrofulaceum ATCC BAA-614] >gb|EFG75472.1|short-chaindehydrogenase/reductase family oxidoreductase [Mycobacteriumparascrofulaceum ATCC BAA-614] 867 868 YP_003871369.13-oxoacyl-[acyl-carrier-protein] reductase (3- LigD ketoacyl-acylcarrier protein reductase) [Paenibacillus polymyxaE681] >gb|ADM70831.1|3-oxoacyl-[acyl-carrier- protein] reductase(3-ketoacyl-acyl carrier protein reductase) [Paenibacillus polymyxaE681] 869 870 ZP_05094873.1 oxidoreductase, short chain LigDdehydrogenase/reductase family [marine gamma proteobacteriumHTCC2148] >gb|EEB78920.1|oxidoreductase, short chaindehydrogenase/reductase family [marine gamma proteobacterium HTCC2148]871 872 ZP_01224235.1 probable oxidoreductase dehydrogenase LigD signalpeptide protein [marine gamma proteobacteriumHTCC2207] >gb|EAS47242.1|probable oxidoreductase dehydrogenase signalpeptide protein [marine gamma proteobacterium HTCC2207] 873 874YP_634033.1 short chain dehydrogenase [Myxococcus LigD xanthus DK1622] >gb|ABF86178.1| oxidoreductase, short chaindehydrogenase/reductase family [Myxococcus xanthus DK 1622] 875 876ABL97174.1 short-chain dehydrogenase/reductase LigD [uncultured marinebacterium EB0_49D07] 877 878 NP_335301.1 short chain dehydrogenase[Mycobacterium LigD tuberculosis CDC1551] >ref|ZP_07413312.2|short-chaintype dehydrogenase/reductase [Mycobacterium tuberculosisSUMu001] >ref|ZP_07668817.1|short-chain type dehydrogenase/reductase[Mycobacterium tuberculosis SUMu010] >ref|ZP_07669069.1|short-chain typedehydrogenase/reductase [Mycobacterium tuberculosisSUMu011] >gb|AAK45115.1| oxidoreductase, short-chaindehydrogenase/reductase family [Mycobacterium tuberculosisCDC1551] >gb|EFO75870.1|short-chain type dehydrogenase/reductase[Mycobacterium tuberculosis SUMu001] >gb|EFP48221.1| short-chain typedehydrogenase/reductase [Mycobacterium tuberculosisSUMu010] >gb|EFP52129.1|short-chain type dehydrogenase/reductase[Mycobacterium tuberculosis SUMu011] 879 880 ZP_01627272.1 short-chaindehydrogenase/reductase SDR LigD [marine gamma proteobacteriumHTCC2080] >gb|EAW39988.1|short-chain dehydrogenase/reductase SDR [marinegamma proteobacterium HTCC2080] 881 882 YP_002774647.1 short chaindehydrogenase [Brevibacillus LigD brevis NBRC 100599] >dbj|BAH46143.1|probable short chain dehydrogenase [Brevibacillus brevis NBRC 100599]883 884 YP_004533909.1 short-chain dehydrogenase/reductase SDR LigD[Novosphingobium sp. PP1Y] >emb|CCA92091.1|short-chaindehydrogenase/reductase SDR [Novosphingobium sp. PP1Y] 885 886ZP_04751842.1 short chain dehydrogenase [Mycobacterium LigD kansasiiATCC 12478] 887 888 ZP_08271356.1 short-chain dehydrogenase/reductaseSDR LigD [gamma proteobacterium IMCC3088] >gb|EGG29327.1|short-chaindehydrogenase/reductase SDR [gamma proteobacterium IMCC3088] 889 890YP_004666338.1 short chain dehydrogenase [Myxococcus LigD fulvusHW-1] >gb|AEI65260.1|short chain dehydrogenase [Myxococcus fulvus HW-1]891 892 YP_001704647.1 putative short chain LigD dehydrogenase/reductase[Mycobacterium abscessus ATCC 19977] >emb|CAM63993.1|Putative shortchain dehydrogenase/reductase [Mycobacterium abscessus] 893 894ZP_07283949.1 cis-2,3-dihydrobiphenyl-2,3-diol LigD dehydrogenase[Streptomyces sp. AA4] >gb|EFL12318.1|cis-2,3-dihydrobiphenyl- 2,3-dioldehydrogenase [Streptomyces sp. AA4] 895 896 YP_002005492.1 hypotheticalprotein RALTA_A1476 LigD [Cupriavidus taiwanensis LMG19424] >emb|CAQ69425.1|putative OXIDOREDUCTASE DEHYDROGENASE[Cupriavidus taiwanensis LMG 19424] 897 898 YP_003543705.1 SDR-familyprotein [Sphingobium japonicum LigD UT26S] >dbj|BAI95093.1|SDR-familyprotein [Sphingobium japonicum UT26S] 899 900 YP_759628.1 short chaindehydrogenase/reductase family LigD oxidoreductase [Hyphomonas neptuniumATCC 15444] >gb|ABI75402.1| oxidoreductase, short chaindehydrogenase/reductase family [Hyphomonas neptunium ATCC 15444] 901 902ZP_03543905.1 short-chain dehydrogenase/reductase SDR LigD [Comamonastestosteroni KF-1] >gb|EED68191.1|short-chain dehydrogenase/reductaseSDR [Comamonas testosteroni KF-1] 903 904 YP_003487191.1 hypotheticalprotein SCAB_14801 LigD [Streptomyces scabiei87.22] >emb|CBG68626.1|putative PROBABLE SHORT-CHAIN TYPEDEHYDROGENASE/REDUCTASE [Streptomyces scabiei 87.22] 905 906 AEG69105.13-oxoacyl-[acyl-carrier-protein] reductase LigD [Ralstonia solanacearumPo82] 907 908 YP_003841993.1 short-chain dehydrogenase/reductase SDRLigD [Clostridium cellulovorans 743B] >ref|ZP_07630916.1|short-chaindehydrogenase/reductase SDR [Clostridium cellulovorans743B] >gb|ADL50229.1|short- chain dehydrogenase/reductase SDR[Clostridium cellulovorans 743B] 909 910 YP_001899010.1 hypotheticalprotein Rpic_1437 [Ralstonia LigD pickettii12J] >gb|ACD26578.1|short-chain dehydrogenase/reductase SDR [Ralstoniapickettii 12J] 911 912 ZP_07965490.1 short chain dehydrogenase[Segniliparus LigD rugosus ATCC BAA-974] >gb|EFV13275.1| short chaindehydrogenase [Segniliparus rugosus ATCC BAA-974] 913 914 NP_250228.1short-chain dehydrogenase [Pseudomonas LigD aeruginosaPAO1] >ref|ZP_01364886.1| hypothetical protein PaerPA_01001998[Pseudomonas aeruginosa PACS2] >ref|YP_002441374.1|putative short-chaindehydrogenase [Pseudomonas aeruginosaLESB58] >ref|ZP_04933207.1|hypothetical protein PA2G_00514 [Pseudomonasaeruginosa 2192] >gb|AAG04926.1|AE004582_4 probable short-chaindehydrogenase [Pseudomonas aeruginosa PAO1] >gb|EAZ57326.1| hypotheticalprotein PA2G_00514 [Pseudomonas aeruginosa2192] >emb|CAW28518.1|probable short-chain dehydrogenase [Pseudomonasaeruginosa LESB58] >gb|EGM16253.1|putative short- chain dehydrogenase[Pseudomonas aeruginosa 138244] 915 916 YP_001020978.1 hypotheticalprotein Mpe_A1784 LigD [Methylibium petroleiphilumPM1] >gb|ABM94743.1|putative oxidoreductase dehydrogenase signal peptideprotein [Methylibium petroleiphilum PM1] 917 918 YP_003745682.1oxidoreductase dehydrogenase [Ralstonia LigD solanacearumCFBP2957] >emb|CBJ43067.1|putative oxidoreductase dehydrogenase[Ralstonia solanacearum CFBP2957] 919 920 ADD82954.1 BatM [Pseudomonasfluorescens] LigD 921 922 ZP_06846575.1 short-chaindehydrogenase/reductase family LigD oxidoreductase [Mycobacteriumparascrofulaceum ATCC BAA-614] >gb|EFG80090.1|short-chaindehydrogenase/reductase family oxidoreductase [Mycobacteriumparascrofulaceum ATCC BAA-614] 923 924 ZP_05041687.1 oxidoreductase,short chain LigD dehydrogenase/reductase family [Alcanivorax sp.DG881] >gb|EDX89108.1| oxidoreductase, short chaindehydrogenase/reductase family [Alcanivorax sp. DG881] 925 926YP_726036.1 hypothetical protein H16_A1536 [Ralstonia LigD eutrophaH16] >emb|CAJ92668.1| conserved hypothetical protein [Ralstonia eutrophaH16] 927 928 ZP_08275744.1 Hypothetical Protein IMCC9480_775 LigD[Oxalobacteraceae bacterium IMCC9480] >gb|EGF30787.1|HypotheticalProtein IMCC9480_775 [Oxalobacteraceae bacterium IMCC9480] 929 930YP_791716.1 putative short-chain dehydrogenase LigD [Pseudomonasaeruginosa UCBPP-PA14] >ref|ZP_06879570.1|putative short-chaindehydrogenase [Pseudomonas aeruginosa PAb1] >ref|ZP_07792770.1|putativeshort- chain dehydrogenase [Pseudomonas aeruginosa39016] >gb|ABJ10717.1| putative short-chain dehydrogenase [Pseudomonasaeruginosa UCBPP-PA14] >gb|EFQ37866.1|putative short-chain dehydrogenase[Pseudomonas aeruginosa 39016] >gb|EGM15719.1|putative short- chaindehydrogenase [Pseudomonas aeruginosa 152504] 931 932 CAQ35702.1oxidoreductase dehydrogenase protein LigD [Ralstonia solanacearum MolK2]933 934 ZP_07966320.1 short chain dehydrogenase [Segniliparus LigDrugosus ATCC BAA-974] >gb|EFV12481.1| short chain dehydrogenase[Segniliparus rugosus ATCC BAA-974] 935 936 YP_002981437.1 hypotheticalprotein Rpic12D_1478 LigD [Ralstonia pickettii 12D] >gb|ACS62765.1|short-chain dehydrogenase/reductase SDR [Ralstonia pickettii 12D] 937938 YP_004685391.1 C alpha-dehydrogenase LigD [Cupriavidus LigD necatorN-1] >gb|AEI76910.1|C alpha- dehydrogenase LigD [Cupriavidus necator N-1] 939 940 ZP_00945631.1 Hypothetical Protein RRSL_01608 LigD [Ralstoniasolanacearum UW551] >ref|YP_002259522.1|oxidoreductase dehydrogenaseprotein [Ralstonia solanacearum IPO1609] >gb|EAP71895.1| HypotheticalProtein RRSL_01608 [Ralstonia solanacearumUW551] >emb|CAQ61454.1|oxidoreductase dehydrogenase protein [Ralstoniasolanacearum IPO1609] 941 942 NP_519890.1 hypothetical protein RSc1769[Ralstonia LigD solanacearum GMI1000] >emb|CAD15471.1|probableoxidoreductase dehydrogenase signal peptide protein [Ralstoniasolanacearum GMI1000] 943 944 ZP_07676733.1 oxidoreductase dehydrogenasesignal LigD peptide protein [Ralstonia sp.5_7_47FAA] >gb|EFP64736.1|oxidoreductase dehydrogenase signal peptideprotein [Ralstonia sp. 5_7_47FAA] 945 946 YP_003752456.1 oxidoreductasedehydrogenase [Ralstonia LigD solanacearum PSI07] >emb|CBJ51176.1|putative oxidoreductase dehydrogenase [Ralstonia solanacearum PSI07] 947948 YP_004533099.1 hypothetical protein PP1Y_AT3242 LigD[Novosphingobium sp. PP1Y] >emb|CCA91281.1|conserved hypotheticalprotein [Novosphingobium sp. PP1Y] 949 950 YP_001564386.1 hypotheticalprotein Daci_3363 [Delftia LigD acidovorans SPH-1] >gb|ABX36001.1|short-chain dehydrogenase/reductase SDR [Delftia acidovorans SPH-1] 951 952YP_004488753.1 short-chain dehydrogenase/reductase SDR LigD [Delftia sp.Cs1-4] >gb|AEF90398.1|short- chain dehydrogenase/reductase SDR [Delftiasp. Cs1-4] 953 954 YP_001188109.1 short-chain dehydrogenase/reductaseSDR LigD [Pseudomonas mendocina ymp] >gb|ABP85377.1|short-chaindehydrogenase/reductase SDR [Pseudomonas mendocina ymp] 955 956ADP99633.1 short-chain dehydrogenase/reductase SDR LigD [Marinobacteradhaerens HP15] 957 958 YP_693638.1 short-chain dehydrogenase/reductasefamily LigD protein [Alcanivorax borkumensisSK2] >emb|CAL17366.1|short-chain dehydrogenase/reductase family[Alcanivorax borkumensis SK2] 959 960 YP_585740.1 short-chaindehydrogenase/reductase SDR LigD [Cupriavidus metalliduransCH34] >gb|ABF10471.1|short-chain dehydrogenase/reductase SDR[Cupriavidus metallidurans CH34] 961 962 YP_003277769.1 short-chaindehydrogenase/reductase SDR LigD [Comamonas testosteroniCNB-2] >gb|ACY32473.1|short-chain dehydrogenase/reductase SDR [Comamonastestosteroni CNB-2] 963 964 ZP_08406457.1 hypothetical protein HGR_11311LigD [Hylemonella gracilis ATCC 19624] >gb|EGI76405.1|hypotheticalprotein HGR_11311 [Hylemonella gracilis ATCC 19624] 965 966YP_003842521.1 short-chain dehydrogenase/reductase SDR LigD [Clostridiumcellulovorans 743B] >ref|ZP_07632312.1|short-chaindehydrogenase/reductase SDR [Clostridium cellulovorans743B] >gb|ADL50757.1|short- chain dehydrogenase/reductase SDR[Clostridium cellulovorans 743B] 967 968 ZP_07043693.1 short-chaindehydrogenase/reductase SDR LigD [Comamonas testosteroniS44] >gb|EFI62855.1|short-chain dehydrogenase/reductase SDR [Comamonastestosteroni S44] 969 970 YP_295629.1 hypothetical protein Reut_A1415[Ralstonia LigD eutropha JMP134] >gb|AAZ60785.1|Short- chaindehydrogenase/reductase SDR [Ralstonia eutropha JMP134] 971 972CBJ37979.1 putative oxidoreductase dehydrogenase LigD [Ralstoniasolanacearum CMR15] 973 974 YP_004155471.1 short-chaindehydrogenase/reductase sdr LigD [Variovorax paradoxusEPS] >gb|ADU37360.1|short-chain dehydrogenase/reductase SDR [Variovoraxparadoxus EPS] 975 976 YP_001353681.1 hypothetical protein mma_1991 LigD[Janthinobacterium sp. Marseille] >gb|ABR91341.1|short-chaindehydrogenase/reductase SDR [Janthinobacterium sp. Marseille]

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, that there are many equivalents tothe specific embodiments described herein that have been described andenabled to the extent that one of skill in the art can practice theinvention well-beyond the scope of the specific embodiments taughtherein. Such equivalents are intended to be encompassed by the followingclaims. In addition, there are numerous lists and Markush groups taughtand claimed herein. One of skill will appreciate that each such list andgroup contains various species and can be modified by the removal, oraddition, of one or more of species, since every list and group taughtand claimed herein may not be applicable to every embodiment feasible inthe practice of the invention. As such, components in such lists can beremoved and are expected to be removed to reflect some embodimentstaught herein. All publications, patents, patent applications, otherreferences, accession numbers, ATCC numbers, etc., mentioned in thisapplication are herein incorporated by reference into the specificationto the same extent as if each was specifically indicated to be hereinincorporated by reference in its entirety.

We claim:
 1. A system for bioprocessing lignin-derived compounds,comprising: a host cell transformed by a vector comprising a recombinantpolynucleotide comprising a nucleotide sequence that encodes apolypeptide having at least 95% identity to SEQ ID NO:541, the aminoacid sequence conserving residues 47-57, 63-76, 100, 101, 104, 107, 111,112, 115, 116, 176, 194, 197, 198, 201, 202, and 206; a lignin-derivedcompound having a beta-aryl ether bond and a molecular weight rangingfrom about 180 Daltons to about 3000 Daltons; and, a solvent in whichthe lignin-derived compound is soluble; wherein, the system functions tocleave the beta-aryl ether bond by contacting the polypeptide with thelignin-derived compound in the solvent.
 2. The system of claim 1,wherein the transformed host cell comprises Azotobacter vinelandii. 3.The system of claim 1, wherein the transformed host cell expresses thepolypeptide of claim 65 in the solvent in which the lignin-derivedcompound is soluble.
 4. A system for bioprocessing lignin-derivedcompounds, comprising: a transformant including a host cell transformedwith a vector comprising a recombinant polynucleotide comprising anucleotide sequence that encodes a polypeptide having at least 95%identity to SEQ ID NO:541, the amino acid sequence conserving residues47-57, 63-76, 100, 101, 104, 107, 111, 112, 115, 116, 176, 194, 197,198, 201, 202, and 206, the transformant expressing the polypeptide; alignin-derived compound having a beta-aryl ether bond and a molecularweight ranging from about 180 Daltons to about 3000 Daltons; and, asolvent in which the lignin-derived compound is soluble; wherein, thesystem functions to cleave the beta-aryl ether bond by contacting thepolypeptide of with the lignin-derived compound in the solvent.
 5. Thesystem of claim 4, wherein the transformant comprises E. Coli.
 6. Thesystem of claim 4, wherein the transformant comprises Azotobactervinelandii.
 7. The system of claim 4, wherein an amino acid substitutionoutside of the conserved residues is a conservative substitution.
 8. Thesystem of claim 4, wherein the transformed host cell expresses thepolypeptide in the solvent in which the lignin-derived compound issoluble.
 9. The system of claim 4, wherein the lignin-derived compoundhas a molecular weight ranging from about 180 Daltons to about 1000Daltons.
 10. The system of claim 4, wherein the solvent environmentcomprises water.
 11. The system of claim 4, wherein the solventenvironment comprises a polar organic solvent.
 12. A system forbioprocessing lignin-derived compounds, comprising: a transformantincluding a host cell transformed with a vector comprising a recombinantpolynucleotide comprising a nucleotide sequence that encodes apolypeptide having SEQ ID NO:541; or conservative substitutions thereofoutside of conserved residues 47-57, 63-76, 100, 101, 104, 107, 111,112, 115, 116, 176, 194, 197, 198, 201, 202, and 206, the transformantexpressing the polypeptide; a lignin-derived compound having a beta-arylether bond and a molecular weight ranging from about 180 Daltons toabout 3000 Daltons; and, a solvent in which the lignin-derived compoundis soluble; wherein, the system functions to cleave the beta-aryl etherbond by contacting the polypeptide with the lignin-derived compound inthe solvent.
 13. The system of claim 12, wherein the transformantcomprises E. Coli.
 14. The system of claim 12, wherein the transformantcomprises Azotobacter vinelandii.
 15. The system of claim 12, whereinthe transformed host cell expresses the polypeptide in the solvent inwhich the lignin-derived compound is soluble.
 16. The system of claim12, wherein the lignin-derived compound has a molecular weight rangingfrom about 180 Daltons to about 1000 Daltons.
 17. The system of claim12, wherein the solvent environment comprises water.
 18. The system ofclaim 12, wherein the solvent environment comprises a polar organicsolvent.
 19. A system for bioprocessing lignin-derived compounds,comprising: a transformant including an Azotobacter vinelandii host celltransformed with a vector comprising a recombinant polynucleotidecomprising a nucleotide sequence that encodes a polypeptide having atleast 95% identity to SEQ ID NO:541, the amino acid sequence conservingresidues 47-57, 63-76, 100, 101, 104, 107, 111, 112, 115, 116, 176, 194,197, 198, 201, 202, and 206, the transformant expressing thepolypeptide; a lignin-derived compound having a beta-aryl ether bond anda molecular weight ranging from about 180 Daltons to about 3000 Daltons;and, a solvent in which the lignin-derived compound is soluble; wherein,the system functions to cleave the beta-aryl ether bond by contactingthe polypeptide with the lignin-derived compound in the solvent.
 20. Thesystem of claim 19, wherein the transformed host cell expresses thepolypeptide of claim 65 in the solvent in which the lignin-derivedcompound is soluble.
 21. The system of claim 19, wherein thelignin-derived compound has a molecular weight ranging from about 180Daltons to about 1000 Daltons.
 22. The system of claim 19, wherein thesolvent environment comprises water.
 23. The system of claim 19, whereinthe solvent environment comprises a polar organic solvent.